Methods and Compositions for Producing Pancreatic Beta Cells

ABSTRACT

Compositions and methods of producing mammalian cell populations that include a high proportion of pancreatic beta cells are described herein. Such cell populations are useful for treatment of diabetes. Also provided are materials and methods for the direct differentiation of stem cells, such as embryonic stem cells, into functional pancreatic beta cells. The disclosure provides the benefit of direct differentiation, which results in the production of functional pancreatic beta cells efficiently and at low cost.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication Nos. 62/197,198 filed Jul. 27, 2015, 62/204,928 filed Aug.13, 2015, and 62/207,200 filed Aug. 19, 2015, each of which applicationsis incorporated herein by reference in its entirety. This application isrelated to PCT application PCT/US2014/048358, filed Jul. 25, 2014, thedisclosure of which is incorporated herein in its entirety by reference.

FIELD

The disclosure generally relates to cell differentiation and methods ofinducing cell differentiation as well as uses of the differentiatedcells in cell therapy and in screens for modulators of celldifferentiation.

INTRODUCTION

Diabetes mellitus type 1 and 2 (T1D, T2D) are diseases characterized byautoimmune destruction or progressive dysfunction and subsequent loss ofinsulin-producing pancreatic beta cells, respectively. For example,Type-1 diabetes results from autoimmune destruction of theinsulin-secreting beta cells within pancreatic islets. Typically itaffects children and young adults. Current methods for management oftype 1 diabetes generally require frequent glucose monitoring andlife-long insulin administration. Current treatments for both types ofpatients with diabetes include regulating blood glucose levels throughinjections of exogenous insulin. While this approach provides reasonablemanagement of the diseases, unwanted risks and long-term complicationspersist due to the inability of tightly maintaining glucose levelswithin a normal physiological range. Complications includelife-threatening episodes of hypoglycemia, as well as long-termcomplications from hyperglycemia resulting in micro- andmacro-angiopathy leading to cardiovascular pathologies and kidneyfailure, as well as neuropathy.

In conjunction with new strategies to induce immune tolerance, thetransplantation of healthy islet and beta cells to replace the lostcells may be a cure for the disease. However, a primary challengeremains—the scarcity of functional, glucose-responsive beta cells. Oneexisting approach to treating diabetes is transplantation of humancadaveric islet preparations into patients. This procedure typicallyresults in better glycemic control, can render patients insulinindependent for prolonged periods of time, and improves overall qualityof life (Shapiro et al, 2000; Barton et al, 2012; Posselt et al, 2010).However, the severe shortage of cadaveric organ donors, requirement forlifelong immunosuppression, and variability between islet preparationshampers the use of islet transplantation as a readily availabletreatment for people with diabetes. Consequently, numerous researchefforts have focused on identifying abundant alternative sources ofsurrogate glucose-responsive insulin-producing cells (Hebrok, 2012;Efrat & Russ, 2012; Nostro & Keller, 2012; Tuduri & Kieffer, 2011;Bouwens et al, 2013; Zhou & Melton, 2008; Pagliuca & Melton, 2013). Oneof the most appealing approaches is the directed differentiation intoinsulin-producing cells from pluripotent human embryonic stem cells(hESC)(D'Amour et al, 2005; Nostro et al, 2011; Guo et al, 2013b; VanHoof et al, 2011; Mfopou et al, 2010; Chen et al, 2009; Xu et al, 2011;Shim et al, 2014) and more recently, induced pluripotent stem cells(Maehr et al, 2009; Shang et al, 2014; Hua et al, 2013).

Researchers have been hopeful that stem cells could provide an unlimitedsource of functional beta cells. Stepwise differentiation conditionshave been proposed that recapitulate developmental signaling and thatpurportedly differentiate pluripotent stem cells through a definitiveendoderm stage all the way into functional pancreatic beta cells(D'Amour, Agulnick et al. 2005; Yasunaga, Tada et al. 2005; Gouon-Evans,Boussemart et al. 2006; Jiang, Shi et al. 2007; Kroon, Martinson et al.2008; Green, Chen et al. 2011). However, use of stem cells as thestarting material for generating pancreatic cells has problems,including lack of availability, potential immunological rejection, andsocial concerns.

Direct beta-cell reprogramming methods could be faster and moreefficient than preparing induced pluripotent stem cells (iPSCs).However, a general approach to converting non-endoderm cells, such asfibroblast cells, across the germ-layer boundary towards anendoderm-beta cell lineage has not yet been developed. Cell typesderived from the endoderm lineage, such as acinar cells or hepatocytes,might be easier to reprogram into a beta cell lineage owing to theirsimilarity to beta cells. However, these methods have not beensuccessfully applied to cell-based therapy or in vivo therapy because ofthe practicality of obtaining useful quantities of starting cells. Inaddition, beta-like cells generated by conventional direct reprogrammingare post-mitotic and have very limited regenerative ability.

Comprehensive knowledge of signaling events and temporal transcriptionfactor (TF) expression patterns during rodent pancreas organogenesis(Pan & Wright, 2011; Seymour & Sander, 2011; Hebrok, 2003; Murtaugh &Melton, 2003) have accelerated the identification of culture conditionsthat allow the generation of pancreatic cell types from humanpluripotent stem cells (hPSC). Early developmental stages, includingdefinitive endoderm, gut tube-like cells and pancreatic progenitors canbe efficiently induced in vitro. Subsequent transitions towardshormone-expressing cells in vitro are less efficient, however, andfrequently lead to the formation of a mixed population of differentpancreatic progenitors and polyhormonal endocrine cells (Guo et al,2013a; Nostro et al, 2011; D'Amour et al, 2006). Such polyhormonal cellsexpress insulin among other hormones, but lack expression of keypancreatic beta cell transcription factors and do not secrete insulin invitro in response to a glucose challenge—the hallmark function of bonafide pancreatic beta cells (Guo et al, 2013a; Nostro et al, 2011;D'Amour et al, 2006). Nonetheless, transplantation of such heterogeneouscultures into surrogate mice results in the formation ofglucose-responsive pancreatic beta cells after several months in vivo(Rezania et al, 2012; Kroon et al, 2008; Szot et al, 2014).

Sophisticated sorting experiments identified progenitor cells expressingPancreatic and Duodenal Homeobox1 TF (PDX1, also known as IPF1) andhomeobox protein NKX6.1 as the source for these functional pancreaticbeta cells (Kelly et al, 2011). While polyhormonal cells have beenidentified in human fetal pancreas, suggesting that they may reflectaspects of the normal embryonic differentiation process (Riedel et al,2011; De Krijger et al, 1992), increasing evidence indicates thathESC-derived polyhormonal cells preferentially give rise to singlehormone-positive alpha-like cells (Rezania et al, 2011). Thus, to fullyreplicate human pancreatic beta cell development in vitro, it isimportant to better understand and accurately recapitulate the sequenceof embryonic signals required for the proper specification of pancreaticbeta cell precursors, rather than alpha cell precursors.

During normal in vivo pancreatic organogenesis, functional pancreaticbeta cells are generated through a step-wise specification processstarting with pancreatic progenitors, identified by the expression ofPDX1 (Herrera et al, 2002). While PDX1⁺ cells can give rise to allpancreatic lineages (Herrera et al, 2002), the subsequent induction ofNKX6.1 in these cells restricts their differentiation potential to onlyendocrine and ductal cells (Schaffer et al, 2010). Endocrinedifferentiation is then initiated in PDX1⁺/NKX6.1⁺ progenitors byshort-lived expression of the basic helix-loop-helix TranscriptionFactor Neurogenin 3 (NEUROG3, also known as NGN3) (Gu et al, 2002).Interestingly, the timing of NEUROG3 expression has been shown to beimportant in promoting the formation of diverse endocrine islet celltypes (Johansson et al, 2007). For example, precocious induction ofendocrine differentiation by forced expression of NEUROG3 in miceresults predominantly in the generation of alpha cells (Johansson et al,2007).

Apparent from the preceding discussion, generation of functionalinsulin-producing pancreatic beta cells for the effective treatment ofdiabetes is a key area of translational research. Stepwisedifferentiation protocols have been devised to guide the differentiationof human embryonic stem cells (hESCs) and, more recently, inducedpluripotent stem cells (iPSCs), into definitive endoderm, primitive guttube endoderm, posterior foregut endoderm, and pancreatic endoderm (PE).These hESC-derived PE cells can mature into functional pancreatic betacells in vivo after prolonged periods following transplantation intoimmunodeficient mice. More recently, improved differentiation protocolshave been described that allow the formation of functional pancreaticbeta cells from hESCs under cell culture conditions. While thesefindings are encouraging, several challenges remain and significantefforts have been directed towards further improvement ofdifferentiation conditions, the expansion of cells at differentprogenitor stages, and the purification of target cell populations inorder to obtain sufficient quantities of functional pancreatic betacells.

A reliable and reproducible source of human pancreatic beta cells is sofar unavailable. Developing a source for greater numbers of humanpancreatic beta cells would have wide application in basic research,drug and toxicology screens, and as a therapeutic product. There is aneed for treatments that provide superior control of glucose metabolismto minimize (e.g., eliminate) long-term complications. There is a needfor the directed differentiation of stem cells (e.g., pluripotent stemcells such as human pluripotent stem cells) into functionalinsulin-producing pancreatic beta cells for the treatment of diabetes.

SUMMARY

The problem of how to obtain or generate significant numbers ofpancreatic beta-like cells has been solved by use of the compositionsand methods described herein. The pancreatic beta-like cells expressinsulin protein and key transcription factors, but only rarely expressother hormones. Compositions and methods are described herein forproducing mammalian cell populations that include a high proportion ofpancreatic beta-like cells and/or mature pancreatic cells. Thepancreatic beta-like cells and mature pancreatic cells so produced maybe particularly useful for treatment of diabetes.

This disclosure describes the efficient generation of such cells invitro. In addition, the disclosed technology offers an improved strategyfor pancreatic beta cell survival upon transplantation by implementingthe disclosed method for direct differentiation of pancreatic beta cellsfrom embryonic stem cells, such as human embryonic stem cells, in thepresence of physiological levels of oxygen.

Cell therapies utilizing functional insulin-producing pancreatic betacells produced from human stem cells hold great promise for thetreatment of diabetes. Current pancreatic differentiation protocolsinduce precocious endocrine differentiation, leading to the formation ofundesired polyhormonal endocrine cells. The disclosure provides asimplified suspension-culture-based differentiation protocol that allowsfor the correct temporal specification of pancreatic and endocrineprogenitors into glucose-responsive pancreatic beta cells in vitro. Thisapproach provides a fast and reproducible supply of functionalpancreatic beta cells (e.g., human pancreatic beta cells) and enablesdetailed investigations into pancreas development and pancreatic betacell biology. Features of the technology disclosed herein may includethe exclusion of commonly used BMP inhibitors during embryonic stemcell-to-pancreatic progenitor cell differentiation, which preventsprecocious endocrine induction. Sequential exposure of foregut cells toretinoic acid followed by combined EGF/KGF treatment of extendedduration establishes highly pure PDX1⁺ and PDX1⁺/NKX6.1⁺ progenitorpopulations, respectively. Precise temporal induction of endocrinedifferentiation in PDX1⁺/NKX6.1⁺ progenitors, but not in PDX1⁺/NKX6.1⁺progenitors, results in the generation of functional pancreatic betacells in vitro. The pancreatic beta cells produced by the disclosedmethods exhibit key features of bona fide human pancreatic beta cells,remain functional after short-term transplantation, and reduce bloodglucose levels in diabetic mice.

Elaborating on the preceding observations, current pancreatic progenitordifferentiation protocols promote precocious endocrine commitment,ultimately resulting in the generation of non-functional polyhormonalcells. Omission of commonly used BMP inhibitors during pancreaticspecification prevents precocious endocrine formation while treatmentwith retinoic acid followed by combined EGF/KGF efficiently generatesboth PDX1⁺ and subsequent PDX1⁺/NKX6.1⁺ pancreatic progenitorpopulations, respectively. Precise temporal activation of endocrinedifferentiation in PDX1⁺/NKX6.1⁺ progenitors produces glucose responsivepancreatic beta cells in vitro that exhibit key features of bona fidehuman pancreatic beta cells, remain functional after short-termtransplantation, and reduce blood glucose levels in diabetic mice. Thus,the disclosed system is a simplified and scalable system that accuratelyrecapitulates key steps of human pancreas development, providing a fastand reproducible supply of functional human pancreatic beta cells.

The methods disclosed herein result in pancreatic beta cells exhibitingimproved function, as evidenced by a rapid and robust response toglucose. The experiments disclosed herein show that more insulin isproduced with vitamin C and BayK-8644 in the culture system, asevidenced by GFP reporter gene expression levels and human C-peptidestaining Thus, the improved functionality of the pancreatic beta cellsproduced using the methods disclosed herein is apparent from bothqualitative and quantitative measures.

In one aspect, the disclosure provides a method of generating aPDX1⁺/NKX6.1⁺ progenitor cell by initially exposing a stem cell (e.g.,an embryonic stem cell such as a human embryonic stem cell or hESC) toan effective amount of a retinoic acid compound, thereby inducingformation of a PDX1⁺ progenitor cell. In some embodiments, the embryonicstem cell is a human embryonic stem cell. In some embodiments, theembryonic stem cell is contacted with a retinoic acid compound in vitro.Embodiments are also contemplated that further comprise not contactingthe embryonic stem cell with a bone morphogenic protein (BMP) inhibitorprior to expression of NKX6.1 by the cell. Omitting BMP from theprotocol for generating PDX1⁺ progenitor cells prevents precociousendocrine differentiation that can lead to nonfunctional polyhormonalcells as schematically shown in FIG. 15. PDX1 progenitor cells can thenbe exposed to an effective amount of epidermal growth factor (EGF),keratinocyte growth factor (KGF), or a combination of EGF and KGF forextended time periods, thereby inducing formation of a PDX1⁺/NKX6.1⁺progenitor cell. In some of these embodiments, the cell expresses NKX6.1prior to the cell contacting at least one of epidermal growth factor andkeratinocyte growth factor. In some of these embodiments, the cellexpresses NKX6.1 prior to contacting epidermal growth factor andkeratinocyte growth factor. In some embodiments, cells are exposed toEGF and KGF simultaneously.

In yet other embodiments, the method further comprises inducing thePDX1⁺/NKX6.1⁺ progenitor cell to express NEUROG3, resulting inproduction of an INS⁺/NKX6.1⁺ beta cell. In some embodiment, the NEUROG3expression is induced by contacting the PDX1⁺/NKX6.1⁺ progenitor cellwith an effective amount of an inhibitor of bone morphogenetic protein,an inhibitor of TGFβ/ALK, or an inhibitor of sonic hedgehog. Embodimentsare contemplated wherein the PDX1⁺/NKX6.1⁺ progenitor cell is contactedby an effective amount of an inhibitor of bone morphogenetic protein andan effective amount of an inhibitor of TGFβ/ALK. In some embodiments,the PDX1⁺/NKX6.1⁺ progenitor cell is contacted by an effective amount ofan inhibitor of bone morphogenetic protein and an effective amount of aninhibitor of sonic hedgehog. In some embodiments, the inhibitor of bonemorphogenetic protein is Noggin and/or the inhibitor of sonic hedgehogis Cyclopamine.

This aspect of the disclosure further provides methods wherein theNEUROG3 expression is induced by exposure of the PDX1⁺/NKX6.1⁺progenitor cell to effective amounts of a TATA-Binding Protein, anActivin receptor-Like Kinase inhibitor, Noggin and Keratinocyte GrowthFactor (KGF or K). In some embodiments, the NEUROG3 expression beginsbefore expression of NKX2.2 is detected. In some embodiments, no morethan 5% of the generated cells are polyhormonal cells. In someembodiments, the INS⁺/NKX6.1⁺ beta cell is responsive to glucose levels.In some of these embodiments, the INS⁺/NKX6.1⁺ beta cell secretes anincreased level of insulin in response to an increased glucose level.

Also provided by this aspect of the disclosure are methods that furthercomprise contacting the PDX1⁺/NKX6.1⁺ progenitor cell with an effectiveamount of vitamin C and/or an effective amount of BayK-8644. Asdisclosed herein, the addition of vitamin C, BayK-8644 or both vitamin Cand BayK-8644 improves the generation of functional pancreatic betacells, also referred to herein as an INS⁺/NKX6.1⁺ beta cells, from stemcells such as embryonic stem cells (e.g., hESCs).

Some embodiments of the methods according to the disclosure are providedwherein the INS⁺/NKX6.1⁺ beta cell does not express a detectable levelof the Ki67 marker.

Another aspect of the disclosure is a method for generating anINS⁺/NKX6.1⁺ beta cell further compromising transplanting theINS⁺/NKX6.1⁺ beta cell into a human. In some embodiments, the human isdiabetic.

Consistent with the foregoing descriptions, an aspect of the disclosureis drawn to a method of producing a pancreatic beta cell from a stemcell comprising: (a) exposing a stem cell to Epidermal Growth Factor(e.g., 10-300 ng/ml Epidermal Growth Factor) and/or Keratinocyte GrowthFactor (e.g., 10-300 ng/ml Keratinocyte Growth Factor) for 12-72 hours[e.g., in DMEM comprising 0.1-5.0 mM glutamine (0.05-2.5X GlutaMAX™;Invitrogen), 0.5-3.0X (e.g., 1X) Invitrogen non-essential amino acids,and 0.5-3.0X (e.g., 1X) B27 supplement (Invitrogen)] under conditionssuitable for cell culture growth, thereby maintaining a progenitor cell;(b) incubating the progenitor cell in a culture medium (e.g., culturemedium comprising DMEM) comprising non-essential amino acids (e.g.,0.1-5X non-essential amino acids, e.g., 1X non-essential amino acids)(e.g., Invitrogen non-essential amino acids), glutamine (e.g., 0.1-5 mMglutamine) or GlutaMax (e.g, 0.05-2.5X GlutaMAX™), heparin (e.g., 2-20μg/ml heparin), cysteine (e.g., 0.2-5 mM cysteine), zinc (e.g., 2-20 μMzinc), ALK inhibitor (e.g., 2-20 μM ALK inhibitor), BMP inhibitorLDN-193189 (e.g., 0.2-2 μM BMP inhibitor LDN-193189), T3 thryroidhormone (e.g., 0.2-5 μM T3 thyroid hormone) and secretase inhibitor XX(e.g., 0.2-5 μM gamma secretase inhibitor XX, e.g., from Calbiochem) toyield a cell in culture; and (c) adding to the cell in culture viamin C(e.g., 10-2000 μM vitamin C) and BayK-8644 (e.g., 0.2-5 μM BayK-8644),thereby producing a functional pancreatic beta cell. In some embodimentsof the method, the stem cell is (a) exposed to 50 ng/ml Epidermal GrowthFactor or 50 ng/ml of Keratinocyte Growth Factor for 12-72 hours in DMEMcomprising 2 mM glutamine (1X GlutaMAX™; Invitrogen), 0.1-5X (e.g., 1X)Invitrogen non-essential amino acids, and 0.1-5X (e.g., 1X) B27supplement (Invitrogen); (b) Incubated in DMEM comprising 0.1-5X (e.g.,1X) Invitrogen non-essential amino acids, 2 mM glutamine (1X GlutaMAX™),10 μg/ml heparin, 1 mM cysteine, 10 μM zinc, 10 μM ALK inhibitor, 0.5 μMBMP inhibitor LDN-193189, 1 μM T3 thyroid hormone and 1 μM gammasecretase inhibitor XX (Calbiochem) to yield a cell in culture; and (c)Adding to the cell in culture 500 μM vitamin C and 2 μM BayK-8644,thereby producing a functional pancreatic beta cell. In someembodiments, the stem cell is exposed to Epidermal Growth Factor orKeratinocyte Growth Factor for 24 hours to 32 days, e.g., 24-48 hours,48 hours to 3 days, 3 days to 5 days, 5 days to 10 days, 10 days to 20days, or 20 days to 32 days. In some embodiments, the stem cell is anembryonic stem cell, such as a human stem cell. Embodiments are alsoenvisioned wherein the stem cell is exposed to epidermal growth factor,or to keratinocyte growth factor, or to both epidermal growth factor andkeratinocyte growth factor.

The methods disclosed herein result in the efficient production ofpancreatic beta cells of improved functionality in exhibiting improvedresponse to glucose. With the addition of vitamin C and Bay K-8644,insulin production is increased, as evident by the strength of thefluorescent signal resulting from expression of GFP in the pancreaticbeta cells produced by the disclosed methods. Thus, the disclosedmethods lead to the production of pancreatic beta cells exhibiting aqualitative and quantitative improvement in functionality. In someembodiments, the functional pancreatic beta cell exhibits aseveral-fold, for example a 1-7-fold, increase in insulin secretion uponstimulation with glucose. Embodiments are also contemplated wherein thepancreatic beta cell is functional immediately upon transplantation, andembodiments are envisioned in which the pancreatic beta cell isfunctional within one week of transplantation. Embodiments are alsocontemplated wherein the pancreatic beta cell remains functional for atleast four weeks, or longer. Yet other embodiments further compriseexposure of the stem cell to an oxygen level no greater than 10% O₂,such as an oxygen level no greater than 5% O₂, or no greater than 4% O₂.In some embodiments, NGN3 and PDX1/NKX6.1 are expressed during theincubating step and the adding step. Embodiments are comprehendedwherein at least 75% of the stem cells differentiate into functionalpancreatic beta cells. The percentage yield of pancreatic beta cells(i.e., 75% pancreatic beta cells) was observed in experiments wherecells are dissociated and allowed to reaggregate in smaller clustersusing Aggrewells (StemCellTechnology) cell culture plates. Controls(undissociated clusters) reveal fewer pancreatic beta cells (about 65%).As shown in mice, Notch signaling is important in endocrinedifferentiation and indicates induction of endocrine differentiationupon disruption of cell-cell contact. It is expected that anotherinfluential factor is the better nutrition supplied to smaller cellclusters. Consistent with the data disclosed herein, an average of about7,000 cells per cluster were generated in ambient oxygen and about 4.000cells per cluster were generated at physiological oxygen levels. Otherembodiments of the method are envisioned wherein the progenitor cell ismaintained for up to 32 days in culture containing Epidermal GrowthFactor and/or Keratinocyte Growth Factor.

Another aspect of the disclosure provides a functional pancreatic betacell produced according to the method described above. In someembodiments of this aspect of the disclosure, the functional pancreaticbeta cell is a human cell. In some embodiments, the cell expresses atleast three pancreatic cell markers selected from the group of humanc-peptide (C-PEP), Chromagranin A (CHGA), transcription factor NKX6.1,transcription factor PDX1, transcription factor PAX6, transcriptionfactor NKX2.2, transcription factor NEUROD1 and transcription factorISL1, without inducing expression of other hormones, e.g., Glucagon(GCG) or Somatostatin (SST), that serve as markers for cells other thanpancreatic beta cells.

Yet another aspect of the disclosure is drawn to a method for treatingdiabetes comprising administering an effective amount of the celldisclosed herein to a diabetic subject, such as a human. One aspect ofthe invention is a method of converting pancreatic endodermal progenitorcells to pancreatic beta-like cells by (a) contacting the pancreaticendodermal progenitor cells with a first composition that includesheparin, zinc salts, a TGF-β inhibitor (e.g., Alk5 inhibitor), a BMP4signaling inhibitor (e.g., LDN-193189), a T3 thyroid hormone, a NotchSignaling Inhibitor (e.g., Compound E), a Ca²⁺ channel agonist (e.g.BayK-8644), vitamin C, and combinations thereof to generate a firstpopulation of partially differentiated pancreatic beta-like cells; (b)contacting the first population of cells with a second composition thatincludes heparin zinc salts, a TGF-β inhibitor (e.g., Alk5 inhibitor), aBMP4 signaling inhibitor (e.g., LDN-193189), a T3 thyroid hormone,cysteine, an anti-oxidant (e.g., vitamin E or a derivative thereof suchas TROLOX®), an Axl kinase inhibitor (e.g., R428), a Ca²⁺ channelagonist (e.g. BayK-8644), vitamin C, and combinations thereof togenerate a second population of pancreatic beta-like cells. In a thirdstep, the second population of cells can be cultured to generate 3Daggregates under low-attachment plates. The pancreatic beta cells thatare generated can be administered to treat diabetes.

The application also provides methods for converting adult cells (e.g.,mammalian starting cells such as fibroblasts) into cells of theendodermal lineage. Once those cells have been converted into endodermalprogenitor cells, they can be differentiated into pancreatic beta cellsusing the method described in the previous paragraph.

Thus, another aspect of the invention is a method that involves: (a)contacting starting mammalian cells with a first composition comprisingan effective amount of a TGFβ family member and a WNT activator, whilethe mammalian cells express pluripotency factors comprising OCT4, SOX2,and KLF4, wherein the effective amount is sufficient to generate a firstcell population comprising definitive endoderm cells and wherein atleast about 5% of the cells in the first population express Sox17 and/orFoxa2, but where the first cell population does not express detectableNANOG; and (b) contacting cells from the first cell population with asecond composition comprising an effective amount of a TGFβ receptorinhibitor, a hedgehog pathway inhibitor, and a retinoic acid receptoragonist to generate a second cell population comprising pancreaticprogenitor cells, wherein at least about 10% of the cells in the secondpopulation express Pdx1, Nkx6.1, Hnf6, or a combination thereof.

Another aspect of the invention is a method that involves contacting anendodermal cell population with an expansion composition that includesgrowth factors, a WNT activator, and a TGFβ receptor inhibitor for atime sufficient to expand cell numbers by at least ten-fold and therebygenerate an expanded population of posterior foregut-like progenitorcells.

The methods can further include administering the second cellpopulation, posterior foregut-like progenitor cells, pancreaticprogenitor cells obtained from the second cell population, functionalpancreatic beta-like cells obtained therefrom, or a combination thereof,to a mammal in need thereof. For example, such a mammal can have type Idiabetes, type II diabetes, or type 1.5 diabetes.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1D illustrate reprogramming of fibroblasts to definitiveendodermal like cells (DELCs) with an iPSC-factor-based lineage-specificreprogramming paradigm. FIG. 1A schematically illustrates two approachesfor reprogramming fibroblasts. Approach-1 (App1) involved transientiPSC-factor expression (induced by doxycycline (Dox) addition to theculture medium) in fibroblasts followed by adding definitive endoderminduction factors Activin A (AA) and lithium chloride (LiCl); Approach-2(App2) involved addition of definitive endoderm induction factors AA andLiCl during the transient doxycycline-induced iPSC-factor expression.FIG. 1B graphically illustrates expression of Sox17 and Foxa2 on Day 12(day 12) after treating fibroblasts by either Approach 1 or Approach 2,where expression levels were assessed by quantitative PCR. The resultsare the average of 3 independent experiments. *P<0.05. FIG. 1Cgraphically illustrates the total number of all the colonies(cross-hatched bar) as well as Sox17⁺/Foxa2⁺ double positive colonies(open bar), where the colonies were generated by using Approach 1 orApproach 2. The results are the average of three independentexperiments. FIG. 1D graphically illustrates the percentage ofSox17⁺/Foxa2⁺ double positive colonies generated by Approach 1 orApproach 2. The results are the average of three independentexperiments.

FIGS. 2A-2G illustrate the proof-of-concept (POC) approach describedherein for reprogramming mouse embryonic fibroblasts (MEFs) intodefinitive endodermal like cells (DELCs), then into pancreaticprogenitor like cells (PPLCs), and then into pancreatic β-like cells(PLCs). FIG. 2A is a schematic diagram of the approach to generatingpancreatic-like cells from fibroblasts by Approach 2. Med-IV containslaminin, nicotinamide, and B27 etc., based on a previous report(Schroeder et al., 2006). FIG. 2B shows images of cells immunostained toidentify expression of definitive endoderm markers Sox17, CXCR4, Foxa2and Cerberus1 in cells on day 12 (D12) of the process outlined in FIG.2A. FIG. 2C graphically illustrates expression of Sox17, Foxa2,Cerberus1, and CXCR4 (left to right panels) in cells treated for twelvedays (DO to D12) by the method shown in FIG. 2A. NGFP1 inducedpluripotent stem cell-derived definitive endoderm like cells (DE) wereused as a positive control. FIG. 2D shows images of cells immunostainedon D18 for pancreatic progenitor markers Pdx1, Hnf6, Pax6, and Nkx6.1(left to right panels). FIG. 2E graphically illustrates expression ofPdx1, Nkx6.1, Hnf6, and Pax6 in cells treated from days D12 to D18 withthe method shown in FIG. 2A. FIG. 2F shows images of cells immunostainedfor pancreatic β cell markers insulin (Ins) and C-peptide (C-PEP) onD27. FIG. 2G graphically illustrates expression of Ins1 and Ins2 incells treated from D18 to D27 with the method shown in FIG. 2A. Theresults in FIGS. 2C, 2E, and 2G are the average of at least threeindependent experiments. *P<0.05, **P<0.01.

FIGS. 3A-3B show that small molecules enhance induction of definitiveendoderm-like cells (DELCs) into pancreatic progenitor like cells(PPLCs). FIG. 3A graphically illustrates expression of Pdx1 and Nkx6.1on day 16 (D16) when the indicated factors of the protocol outlined inFIG. 4A are employed. Cells were treated with indicated small moleculesfrom day 12 to day 16 (D12 to D16). The results shown are the average of3 independent experiments. *P<0.05. FIG. 3B shows images of cellsimmunostained on day 16 (D16) for Pdx1 and Nkx6.1 after treatment withthe indicated conditions.

FIGS. 4A-4F illustrates identification of novel small moleculeconditions that enhanced generation of pancreatic progenitor-like cellsthat are readily converted in the pancreatic beta cells. FIG. 4A is aschematic diagram of the advanced approach where additional smallmolecules (Bix (Bix-01294), and pVc (2-Phospho-L-ascorbic acid trisodiumsalt), A83, and/or SB (SB203580)) are employed that significantlyimprove induction of the pancreatic progenitor-like phenotype. Asillustrated, mouse embryonic fibroblasts (MEFs) are redirected to formIntermediate Cells (IMCs) by exposure to doxycycline (which inducespluripotency factor expression), Activin A, LiCl, Bix (Bix-01294), andpVc (2-Phospho-L-ascorbic acid trisodium salt) between days 0-6. Furtherexposure of the IMCs to Activin A, LiCl, and pVc directs the cells todifferentiate into definitive endoderm-like cells (DELCs) by day 12.When the DELCs are exposed to retinoic acid (RA), A83-01, LDE225 andpVc, they differentiate into pancreatic progenitor-like cells (PPLCs) byday 16, which can be further differentiated into insulin-producingpancreatic-like cells (PLCs) by day 25. FIG. 4B shows images of cellsimmunostained on Day 16 for pancreatic progenitor markers, Nkx6.1, Pdx1,Sox9, Pax6, and Hnf6. Cells were treated with the combination of foursmall molecules (RA, A83-01, LDE225 and pVc) from D12 to D16 to generatethe cells shown in FIG. 4B. FIG. 4C graphically illustrates expressionof the indicated markers at days 12, 14 and 16 during the pancreaticprogenitor induction process as detected by quantitative PCR. Theresults are the average of 3 independent experiments. *P<0.05, **P<0.01.FIG. 4D shows images of cells immunostained on D16 for Pdx1 and Nkx6.1.FIG. 4E graphically illustrates Pdx1⁺/Nkx6.1⁺ colony numbers on D16after treatment of cells with the indicated conditions. 4000 cells wereseeded into each well of a 24-well plate on DO. The results are theaverage of 3 independent experiments. *P<0.05. FIG. 4F shows images ofcells immunostained on day 12 (D12) for expression of Sox17, Foxa2,Cerberus1 and CXCR4. Bix (Bix-01294) at 1 μM was added from DO to D6 andpVc (2-Phospho-L-ascorbic acid trisodium salt) at 280 μM was added tothe cells from DO to D12 before the images shown in FIGS. 4B, 4D, and 4Fwere taken.

FIGS. 5A-5D show that pancreatic progenitor-like cells can bedifferentiated into pancreatic-like cells. FIG. 5A shows images of cellsimmunostained on day 25 (D25) to show which cells express Pdx1 andinsulin, after SB (SB203580) at 50 μM was added from D16 to D25, and pVc(2-Phospho-L-ascorbic acid trisodium salt) at 280 μM was added from D16to D25. FIG. 5B graphically illustrates expression of Ins1, Ins2, Pdx1and Nkx6.1 during the process as assessed by quantitative PCR. Theresults are the average of 3 independent experiments. *P<0.05, **P<0.01.FIG. 5C shows images of cells immunostained on day 25 (D25) to showwhich cells express the pancreatic cell markers insulin, Glucagon,Somatostatin, Amylase and Nkx6.1. FIG. 5D graphically illustratesinsulin release on day 25 by cells treated as described herein. Theresults are the average of 4 independent experiments. *P<0.05. Asindicated, the pancreatic cells produced by the methods described hereinexpress insulin in a glucose dose-responsive manner.

FIGS. 6A-6D shows that induced pluripotent stem cells were not generatedduring the reprogramming process. FIG. 6A graphically illustrates geneexpression of the indicated markers as a function of time during thereprogramming process, as assessed by quantitative PCR. The expressionlevels of pluripotency genes, Nanog and Rex1; of definitive endodermmarker genes, Sox17 and Foxa2; of pancreatic progenitor markers Pdx1 andNkx6.1; and of pancreatic beta cell markers Ins1 and Ins2 are shown.NGFP1 iPS cells (iPS) are used as positive control for Nanog and Rex1expression. Definitive endoderm like cells (DE) derived from NGFP1 iPScells were used as a positive control for Sox17 and Foxa2 expression.The results are the average of four independent experiments. *P<0.05,**P<0.01. FIG. 6B shows images of cells tracing the direct reprogrammingprocess over time from mouse embryonic fibroblasts (MEF; at day 0) todefinitive endodermal like cells (DELCs) at day 12. Bright-field andfluorescence images were taken at the indicated times. Representativeimmunostaining of Sox17 and Foxa2 on Day 12 is also shown (last panel,D12). Secondary MEF cells that were cultured in Med-I with Dox and LIFfor 17 days are shown as a positive control (first panel, Ctrl). FIG. 6Cshows images of cells tracing the induction/differentiation process ofDELC to PPLC. The last image shows representative immunostaining ofcells to identify Pdx1 and Nkx6.1 expression on Day 16. FIG. 6D showsimages of cells tracing the induction/differentiation process of PPLC topancreatic like cells. The right-most image shows representativeimmunostaining of cells exhibiting Ins and Glu expression on Day 25.

FIGS. 7A-7D shows that pancreatic progenitor like cells can regulateglucose levels and insulin secretion in vivo. FIG. 7A graphicallyillustrates blood glucose levels of normal mice and STZ-induced type Idiabetic mice transplanted with MEF or PPLC cells (or controls nottransplanted with cells) during the eight weeks (0 W, 1 W, etc.)following transplantation. Under anesthesia, diabetic mice received arenal subcapsular transplant of about 3×10⁶ pancreatic progenitor likecells (n=14; filled diamond symbols) or 3×10⁶ secondary MEF cells (n=10;filled square symbols). Untreated normal mice (n=4, lower line with Xsymbols) and mice treated with STZ only (n=4; filled triangle symbols)were used as controls. *P<0.05, **P<0.01. FIGS. 7B and 7C show images ofkidney sections from mice engrafted with PPLCs, where theimmunofluorescence identifies sites of gene expression. The C regionsshow images of regions containing transplanted cells, while the Kregions identify kidney tissues. FIG. 7D graphically illustrates seruminsulin levels in the serum of animals from each experimental or controlgroup. The group of mice transplanted with MEF cells was set as control.**P<0.01.

FIGS. 8A-8G illustrate direct conversion of human fibroblasts intodefinitive endodermal progenitor cells. FIG. 8A is a schematic diagramof the procedure used to convert human fibroblasts into definitiveendodermal progenitor cells (cDE cells) by combining non-integratingepisomal reprogramming plasmids with specific initiation and conversionconditions. FIG. 8B shows bright-field images of control fibroblasts anda definitive endodermal progenitor cell (cDE) colony at day 21. Scalebar, 20 μm. FIG. 8C illustrates immunofluorescence staining ofrepresentative definitive endodermal progenitor cell (cDE) colonies atday 21 for the endodermal progenitor markers SOX17 and FOXA2. Scale bar,20 μm. FIG. 8D graphically illustrates the colony number of number ofFOXA2 positive colonies scored at day 28 after treatment of cells withvarious small molecule factors. Small molecules sodium butyrate (NaB),Parnate (Par), RG108 (RG), CHIR99021 (CHIR), and5′-N-ethylcarboxamidoadenosine (NECA) added to the basal condition tofurther enhance endodermal reprogramming efficiency. Data represent thenumber of FOXA2 positive colonies scored at day 28 (Mean values±standarderror (s.e.m.) of three experiments). FIG. 8E graphically illustratesexpression levels (relative quantity, RQ, values) of endodermal genesSOX17 and FOXA2, and endogenous pluripotent genes OCT4 (endoOCT4) andNANOG (endoNANOG) during the conversion process. Human ESCs (hES) servedas a control. Mean values±s.e.m. of expression for the indicated genewere normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)expression for day 28 (d28) definitive endodermal progenitor cellcultures and hESCs, respectively (n=3 experiments). FIG. 8F graphicallyillustrates the percentage of cells expressing human pluripotent surfacemarker TRA-1-60 and endodermal marker FOXA2 during the conversionprocess as assessed by fluorescence-activated cell sorting. Human ESCs(hES) served as control for comparison. FIG. 8G shows images ofdefinitive endodermal progenitor cell colonies at day 28, illustratingexpression of early endodermal progenitor markers SOX17 and FOXA2 (upperpanels, lighter areas), but not of the primitive gut tube marker HNF4A,posterior foregut marker HNF6, and pluripotency marker NANOG (middlepanels) where only DAPI staining of nuclei is visible. Note that hESCserved as positive control for NANOG staining (right panel in the middlerow). Control fibroblasts (lower panels) do not express any markeranalyzed. Scale bar, 20 μm.

FIGS. 9A-9P illustrate the specificity, expansion and characterizationof human posterior foregut-like progenitor cells. FIG. 9A is a schematicrepresentation of culture conditions for the amplification of definitiveendodermal progenitor cells (cDE cells) that resulted in furtherspecification into expandable posterior foregut-like progenitor cells(cPF cells). FIG. 9B illustrates that improved culture conditions allowamplification of cDE/cPF cell colonies. Mean values±s.e.m. representthree experiments. FIG. 9C shows images of colonies after 4 passages inimproved expansion media indicating that the cells exhibit specificationtowards cPF cells as detected by immunofluorescence analysis. Scale bar,20 μm. FIG. 9D is a schematic diagram of the process for cPF expansion.FIG. 9E graphically illustrates growth curves of cPF cells. Note thatthe cPF cell number increases at least a trillion fold. FIG. 9Fgraphically illustrates the effects of four media supplements, EGF,bFGF, A83-01, and CHIR99021 upon cell growth. As shown, the combination(“ALL”) of these factors improves cPF cell growth and self-renewal. FIG.9G shows a bright field image of established cPF cells illustratingepithelial colony morphology. Scale bar, 20 μm. FIG. 9H illustratesexpression of SOX17, FOXA2, HNF4α, HNF6, SOX9, and PDX1 in cPF cells atpassage 15 as detected by immunofluorescence staining Scale bar, 20 μm.FIG. 9I graphically illustrates enrichment of transcripts for SOX17,FOXA2, HNF1A, HNF1B, HNF4A, HNF6, SOX9, and PDX1 transcripts, but notSOX1, BRY, OCT4 and NANOG in p15 cPF cells as detected by qPCR analysis.Mean values±s.e.m. were normalized to GAPDH relative to controlfibroblasts. (n=3 experiments). FIG. 9J graphically illustrates qPCRanalysis for the presence of episomal reprogramming vectors inestablished cPF cells (cPF p17) compared to fibroblasts 4 days afterelectroporation with episomal plasmids, serving as positive control(pos. CTR). Note that episomal vectors were undetectable by this methodin cPF cells. Mean values±s.e.m. are shown (n=3 experiments). FIG. 9Kshows images of kidney tissues after transplantation of hESC-deriveddefinitive endodermal cells and cPF cells under the kidney capsule ofimmune deficient mice. None of the cPF cell grafts result in tumorformation, even after prolonged periods of up to 12 weeks in vivo (n=10mice), while hESC-derived endodermal cell grafts (n=4 mice) resulted intumorigenic structures with big cysts and increased graft size alreadyafter 7 weeks in vivo. FIG. 9L shows images of cPF cell graftsexhibiting epithelial structures that express E-cadherin, HNF4α, PDX1,SOX9, and pan-cytokeratin as detected by immunofluorescence analysisHuman nuclear antigen (HuNu) identifies the human cell origin. Scalebar, 20 μm. FIG. 9M shows expression of transcription factor HNF4a(light areas; green in the original) and albumin (ALB; light areas, redin the original) by p5 and p14 cPF cells that were differentiated intohepatocytes. Nuclei are visualized by DAPI staining Scale bar, 50 μm.FIG. 9N shows a representative image of a haematoxylin and eosin stainedcPF graft section. Stars indicated multiple epithelial structures. Scalebar, 50 μm. FIG. 9O shows expression of SOX17, FOXA2, HNF4A, HNF6, SOX9and PDX1 by cPF cells obtained from human adult fibroblasts as observedby immunofluorescence analysis. Nuclei are visualized by DAPI staining.Scale bar, 50 μm. FIG. 9P shows expression of PDX1 and NKX6.1 by cPEcells obtained from human adult fibroblasts as observed byimmunostaining Nuclei were visualized by DAPI staining Scale bar, 50 μm.

FIGS. 10A-10L illustrate the differentiation of human posteriorforegut-like progenitor cells (cPF cells) into expandable pancreaticendodermal progenitor cells with the ability to mature into functionalbeta-like cells in vivo. FIG. 10A is a schematic diagram of a processfor the differentiation of cPF cells into pancreatic endodermalprogenitor cells (cPE cells). FIG. 10B and FIG. 10C illustrateexpression (lighter areas) of pancreatic endodermal markers FOXA2, SOX9,HNF6, PDX1 and NKX6.1 in p1 cPE cells as detected by immunofluorescencestaining Scale bar, 20 μm. FIG. 10D graphically illustrates thepercentage of cells that express PDX1 and NKX6.1 in p1 cPE cellscompared to control (Isotype CTR) as detected by flow cytometry. FIG.10E is a schematic diagram illustrating procedures for the expansion ofcPE cells. FIG. 10F graphically illustrates growth of cPE cells overtime. Note that cPE cell number increases at least two hundred millionfold. FIG. 10G illustrates expression of pancreatic endodermal markersPDX1 and NKX6.1 in p12 cPE cells as detected by immunofluorescencestaining Scale bar, 20 μm. FIG. 10H graphically illustrates thepercentage of p12 cPE cells that express PDX1 and NKX6.1 as detected byflow cytometry. FIG. 10I graphically illustrates enrichment of NKX2.2,NKX6.1, PDX1, FOXA2, HNF4A, HNF6, HLXB9, PTF1A, and NGN3 transcripts,while SOX17 expression is down regulated, in cPE cells relative to cPFcells. Gene expression was detected by qPCR analysis. Mean expressionvalues±s.e.m. of the indicated genes were normalized to GAPDH and (n=3experiments). FIG. 10 J graphically illustrates that detectable levelsof human c-peptide (as detected by ELISA analysis) are present in theserum of 67% of mice bearing cPE cell grafts for 15 weeks and within 1hour after glucose challenge. Moreover, human C-peptide levels as wellas the percentage of mice exhibiting detectable levels of humanC-peptide increase over time. Numbers on top of each bar indicate humanC-peptide positive mice out of total mice assayed. FIG. 10K graphicallyillustrates human C-peptide levels before and after glucose challenge ofmice bearing cPE cell grafts for 23-24 weeks as detected by ELISAanalysis. As shown, the mice exhibit a functional response to glucoseadministration. The line at about 15 pM human C-peptide identifies thelower detection limit of the ELISA assay. The p-value was calculatedusing a two tailed students t-test. FIG. 10L illustrates co-expressionof insulin (INS) and the beta cell transcription factors PDX1 and NKX6.1but not of the hormone Glucagon (GCG) as detected by immunofluorescenceanalysis of 15-week-old cPE cell graft sections. Scale bar, 20 μm.

FIGS. 11A-11H illustrate maturation of cPE cells into humaninsulin-producing, glucose-responsive pancreatic beta-like cells (cPB)in vitro. FIG. 11A is a schematic diagram of the strategy employed tomature pancreatic endodermal progenitor cells (cPE cells) intopancreatic beta-like cells (cPB cells) in vitro. FIG. 11B illustratesexpression of PDX1 and C-peptide in beta-like cells generated with basalpancreatic differentiation conditions in vitro as detected byimmunofluorescence staining Scale bar, 20 μm. Basal pancreaticdifferentiation media contains A83-01 (A83), Nicotinamide (NIC),Forskolin (FSK), Dexamethasone (DEX) and Exendin-4 (Ex-4). FIG. 11Cgraphically illustrates the effects of several small molecules, CompoundE (C-E), Vitamin C (Vc), and BayK-8644 (BayK) on the percentage ofC-peptide positive cells. Note that combined treatment of all moleculesresults in an additive effect, further increasing the percentage ofC-peptide positive cells. (n=3 experiments) FIG. 11D illustratesexpression of several genes in converted pancreatic beta-like cells (cPBcells) as detected by immunofluorescence analysis. The cPB cells weregenerated with the improved pancreatic maturation conditions. Many ofthe insulin (INS) positive cells co-express key beta cell transcriptionfactors including, PDX1, NKX6.1, NKX2.2, and NeuroD, but only rarelyco-express endocrine progenitor marker NGN3 and the endocrine hormones,Glucagon (GCG) and Somatostatin (SST). Scale bar, 20 μm. FIG. 11Egraphically illustrates human C-peptide levels (a marker for insulinrelease) during in vitro glucose stimulation of insulin secretion (GSIS)assays (n=7 cell cultures of 4 experiments). As shown, the cPB cellsrelease insulin in response to physiological levels of glucose.Depolarization by higher KCl concentration further increased insulinsecretion. Insulin release was measured by human specific C-peptideELISA assay. The line at about 10-15 pM human C-peptide identifies thelower detection limit of the ELISA assay. The p-value was calculatedusing a two tailed students t-test. FIG. 11F illustrates the percentageof cPB cells converted from fibroblasts that express endodermal andpluripotency markers (PDX1, NKX6.1, NKX2.2) in as detected by flowcytometry of fluorophores A-488 and A-647. The first panel showsfluorescence by the secondary antibody only. FIG. 11G illustratesexpression of several genes in cPB cells. Many of the insulin (INS)positive cells co-express key beta cell transcription factors PDX1,NKX6.1, NKX2.2, and NeuroD, but only rarely co-express endocrineprogenitor marker NGN3. Scale bar, 20 μm. FIG. 11H shows highmagnification micrographs of cPB cells. As shown, insulin (INS) andC-peptide staining is co-localized, excluding insulin uptake from themedia as an explanation for C-peptide staining Scale bar, 20 μm.

FIG. 12A-12H illustrate improved maturation of cPE cells intoinsulin-producing, glucose-responsive cPB cells in vitro. FIG. 12A is aschematic diagram of the improved approach (protocol 2) employed tomature cPE cells into cPB cells in vitro. The improved protocol 2consists of two candidate factors, Vitamin C (Vc) and BayK-8644 (BayK),identified by a chemical screen in conjugation with other factorsdescribed herein. FIG. 12B shows that addition of Vitamin C (Vc), andBayK-8644 (BayK) increases mRNA levels of INSULIN (INS) gene indifferentiated cPB cultures at day 21. n=3 experiments. Statisticalsignificance was calculated using two-tailed student's t-test, comparedto DMSO controls. **P<0.01. FIG. 12C shows the immunofluorescencedetected in cPB cells generated with the improved pancreatic maturationconditions. Many of the C-peptide (C-pep) positive cells co-express keybeta cell transcription factors, PDX1 and NKX6.1, but only rarelyco-express other endocrine hormones, Glucagon (GCG) and Somatostatin(SST). Scale bar, 50 μm. FIG. 12D graphically illustrates flowcytometric analysis of cPB cells for human C-peptide (C-pep), Glucagon(GCG) and Somatostatin (SST). FIG. 12E graphically illustrates thepercent of total cells that express a C-pep alone or with GCG or SST.n=3 experiments. FIG. 12F graphically illustrates the amount ofC-peptide detected in an in vitro glucose stimulated insulin secretion(GSIS) assay (n=7 cell cultures of 3 experiments). These resultsdemonstrate that cPB cells release insulin in response to physiologicallevels of glucose. Depolarization by higher KCl concentration furtherincreased insulin secretion. Note that insulin release was measured byhuman specific C-peptide ELISA assay. P-value was calculated using atwo-tailed student's t-test. FIG. 12G is a schematic diagram of alentiviral Insulin gene promoter reporter construct where the Insulingene promoter is operably linked to an mCherry coding region. Pancreaticendodermal progenitor (cPE) cells were infected with the lentiviralconstruct before differentiation in to pancreatic beta-like (cPB) cells.After conversion of the cPE to cPB cells, the cPB cells were sorted,those expressing mCherry were collected, and the collected cPB cellswere evaluated for expression of variety of genes. FIG. 12H graphicallyillustrates expression of the following genes in mCherry-expressing cPBcells (left bar) compared to primary human islet cells (right bar): INS,MAFA, PDX1, NKX6.1, NKX2.2, PCSK1, KIR6.2, SUR1, NEUROD1, RFX6, PAX6,GCK, SLC30A, and UCN3 8. P-values were calculated using a two-tailedstudent's t-test. *P<0.05, **P<0.01.

FIG. 13A-13D illustrate that transplanted pancreatic beta-like (cPB)cells generated by protocol 2 remain functional and protected mice fromchemically induced diabetes. FIG. 13A is a schematic diagram of thetransplantation of cPB into immunodeficient mice. FIG. 13B graphicallyillustrates the amount of human C-peptide as detected by ELISA analysisof serum from fasted and glucose challenged mice two months posttransplantation with either fibroblasts (Fib) or cPB. cPB graft bearingmice exhibit significant higher levels of circulating human C-peptide inserum after a glucose bolus, indicating that transplanted cPB cellsremain functional in vivo. Mice transplanted with Fib controls do notexhibit circulating human C-peptide. n=12 mice for cPB and n=5 mice forFib. P-value was calculated using a two-tailed student's t-test. FIG.13C shows the immunofluorescence of 2-month-old cPB cell grafts,illustrating the co-expression of C-peptide (C-pep) and the beta celltranscription factors PDX1 and NKX6.1 but not of the hormones Glucagon(GCG) and Somatostatin (SST). Scale bar, 50 μm. Data shown arerepresentative of 2 mice. FIG. 13D graphically illustrates fed bloodglucose levels of mice bearing cPB grafts (ovals) with circulating humanC-peptide levels above 200 pM after glucose stimulation and controlfibroblasts (diamonds) are shown. Mice were treated with the mousespecific beta cell toxin Streptozotocine (STZ) to ablate endogenous betacells. Unilateral nephrectomy of cPB graft bearing mice 5 weeks afterSTZ treatment resulted in a rapid rise in blood glucose levels, directlydemonstrating euglycemic control due to cPB grafts after STZ treatmentin these mice. n=6 mice for cPB and n=6 mice for Fib. P-value wascalculated using a two-tailed student's t-test. *P<0.05.

FIG. 14A-14D further illustrate the characteristics of functionalpancreatic beta-like cells (cPB) from human adult fibroblasts generatedaccording to FIG. 12A. FIG. 14A shows expression of C-peptide (C-pep),Glucagon (GCG) and Somatostatin (SST) by cPB cells (obtained from adulthuman fibroblasts) as detected by immunofluorescence analysis of Scalebar, 20 μm. FIG. 14B shows expression of human C-peptide (C-pep),Glucagon (GCG) and Somatostatin (SST) by cPB cells as detected by flowcytometric analysis. FIG. 14C graphically illustrates the percent of cPBcells (obtained from adult human fibroblasts) that express one or two ofhuman C-pep and GCG or SST as detected by flow cytometry. FIG. 14Dgraphically illustrates the amount of human C-peptide expressed asdetected by in vitro glucose stimulated insulin secretion (GSIS) assays(n=7 cell cultures of 4 experiments). As shown, the cPB cells (fromadult human fibroblasts) release insulin in response to physiologicallevels of glucose. Depolarization by higher KCl concentration furtherincreased insulin secretion. Note that insulin release was measured byhuman specific C-peptide ELISA assay. P-value was calculated using a twotailed student's t-test.

FIGS. 15A-15H. Pancreatic differentiation of hESCs using a large-scaleculture system results in two distinct subsets of insulin-producingcells. FIG. 15A: Schematic outlining the differentiation methodemployed. R=Retinoic acid, C=Cyclopamine, N=Noggin, E=Epidermal growthfactor, K=Keratinocyte growth factor, T=TBP, and A=ALK inhibitor. FIG.15B: Micrograph of MEL1INS-GFP cell clusters after 17 days ofdifferentiation demonstrating strong GFP expression (GFP expression inwhite). Scale bar=200 μm. FIG. 15C: Flow cytometric analysis at day 20of differentiation showing 41.5% of all cells expressing GFP under thecontrol of the endogenous insulin promoter. FIG. 15D: Quantification byflow cytometry of the average percentage of GFP cells withindifferentiated cultures after 19-24 days. n=7. Values areaverage±standard deviation (SD). FIG. 15E: Flow cytometric analysis ofintracellular human-specific C-peptide (C-PEP) and insulin (INS) showscomparable percentages of C-PEP and INS positive cells. FIG. 15F:Immunofluorescence staining for C-PEP and glucagon (GCG), and flowcytometric quantification of GCG⁺/C-PEP⁺ (red gate), GCG⁻/C-PEP⁺ (blackgate) populations at days 13 and 19 of differentiation. FIG. 15G:Immunofluorescence staining for C-PEP and NKX6.1, and flow cytometricquantification of NKX6.1⁺/INS⁺ (green gate) and NKX6.1⁻/INS⁺ (red gate)populations at days 13 and 19 Immunofluorescence insets show twodistinct phenotypes for C-PEP cells (NKX6.1⁺ and NKX6.1⁻). A robustINS/NKX6.1 double positive population is only detected at day 19. FIG.15H: Transmission electron microscopy of day 20 clusters. Cells containboth secretory vesicles with electron dense cores surrounded by electronlight halos (green box), akin to bona fide pancreatic beta cellvesicles, as well as other granules similar to those found in non-betapancreatic cells (red boxes).

FIGS. 16A-16D. Published protocols result in precocious endocrinedifferentiation by NEUROG3 activation. FIGS. 16A-C: Analysis of keypancreatic progenitor markers in clusters differentiated as outlined inFIG. 15A. R=Retinoic acid, C=Cyclopamine, N=Noggin, E=Epidermal growthfactor, K=Keratinocyte growth factor, T=TBP, and A=ALK inhibitor. Datashown are representative of two independent experiments. FIG. 16A: PDX1,INS, GCG, NEUROG3, and NKX6.1 protein expression was assessed by wholemount staining of differentiated clusters at indicated time points. Noteprecocious expression of the endocrine marker NEUROG3 in the absence ofNKX6.1 protein at days 6-9. FIGS. 16B and 16C: Flow cytometricquantification of PDX1⁺ (orange gate), PDX1⁺/NKX6.1⁺ (blue gate),INS⁺/NKX6.1⁺ (green gate), and INS⁺/NKX6.1⁻ (red gate) cells atindicated time points. FIG. 16D: qPCR analysis of NGN3 and NKX2.2transcripts at day 8 of differentiation employing RCN (Retinoic acid(R), Cyclopamine (C), and Noggin (N)) or R with two differentconcentrations of vitamin C (Vit. C) treatment for 3 days or withouttreatment. Data are shown as the average±standard error, relative to RCNand normalized to GAPDH. (n=three independent experiments, technicalduplicates).

FIGS. 17A-17C. Defining the temporal activities of individual signalingfactors to efficiently generate PDX1⁺ and PDX1⁺/NKX6.1⁺ pancreasprogenitor populations while preventing precocious induction ofendocrine differentiation. FIGS. 17A-C: Pancreatic progenitor markerexpression at day 9.5 after treatment with conventional differentiationfactors alone or in different combinations. Treatments consisted ofcombinations of Cyclopamine (C), Noggin (N), and retinoic acid (R)during days 6-8, followed by subdivision of each condition into threetreatment groups during day 9-9.5. Group 1) continuation of day 6-8treatment; Group 2) treatment with EGF and KGF (EK); Group 3) treatmentwith EGF, KGF, and Noggin (EKN). The condition selected for furtherstudies, ‘10’, is marked with a green box. Data shown arerepresentatives of results obtained in two independent experiments. FIG.17A: Table detailing 18 different culture conditions that wereevaluated. FIG. 17B: Quantification of PDX1 (orange gate) and NKX6.1(blue gate) protein expressing cells in individual conditions after 9.5days of differentiation. FIG. 17C: NKX6.1 and NEUROG3 protein expressionassessed by whole mount staining of differentiated clusters at 9.5 days.Note robust NEUROG3 expression in all clusters exposed to N (conditions3, 6, 9, and 12-18).

FIGS. 18A-18C. Recapitulating human pancreas organogenesis to generateendocrine progenitors. FIG. 18A: Schematic outlining a simplifieddifferentiation strategy for the controlled, step-wise generation ofpancreatic progenitor cell types. FIG. 18B: Time-course flow cytometricanalysis illustrates the efficient generation of PDX1⁺ progenitor(orange gate) and PDX1⁺/NKX6.1⁺ progenitor (blue gate) populations. Datafrom one of three independent experiments with similar results areshown. FIG. 18C: Immunofluorescence analysis of sections fromdifferentiated clusters at indicated time points stained for humanNKX2.2 (green) and NEUROG3 (red). Insets show NEUOG3/NKX2.2 doublepositive cells. Data from one of three independent experiments withsimilar results is shown.

FIGS. 19A-19F. Efficient generation of PDX1⁺/NKX6.1⁺ pancreaticprogenitor cells prior to endocrine induction results in pancreatic betacells. FIG. 19A: Schematic outlining a simplified differentiationstrategy for the controlled, step-wise generation of pancreaticprogenitor and subsequent endocrine cell types. GFs=growth factors. FIG.19B: Micrographs of differentiated clusters at day 19 under lightmicroscopy (left picture) or fluorescent microscopy showing prominentGFP expression (right picture; GFP expression shown in white). FIG. 19C:Quantification of the percentage of human C-peptide positive cells atday 19-21. Values are average ±SD. n=7 independent experiments. FIG.19D: Immunofluorescence stainings of differentiated clusters at day 20for insulin (INS), PDX1, NKX6.1, NKX2.2 and glucagon (GCG). One of fourexperiments with similar outcome is shown. FIG. 19E: Representative flowcytometry plots depicting co-expression of pancreatic markers PDX1,NKX6.1, NKX2.2, ISL1, NEUROD1, PAX6, Chromogranin A (CHGA), and GCG withhuman C-peptide at indicated time points. Black gates mark percentage oftotal cells positive for indicated marker on ‘Y’ axis. Green gates markpercentage of double positive pancreatic beta cells. The red gate markspercentage of INS⁺/GCG⁺ bihormonal cells. FIG. 19F: Flow cytometricquantification of C-peptide positive pancreatic beta cells co-expressingmarkers in ‘D’. A high percentage of pancreatic beta cells co-expressall genes usually found in pancreatic beta cells, but not the hormoneGCG. Values are average ±SD. n=4 for PDX1, n=19 for NKX6.1, n=4 forNKX2.2, n=9 for ISL1, n=9 for NEUROD1, n=5 for PAX6, n=6 for CHGA, andn=5 for GCG. Analysis was performed at days 15-21 of differentiation.

FIGS. 20A-20B. hESC derived pancreatic beta cells are post-mitotic. FIG.20A: Proliferation of C-peptide⁺ pancreatic beta cells and C-peptidenegative cell populations at days 18-20 was determined by co-stainingwith the proliferation marker Ki67. FIG. 20B: Immunofluorescencestaining of differentiated clusters at day 20 for the proliferationmarker Ki67 and human insulin (INS). Representative data from one ofthree experiments with similar results are shown.

FIGS. 21A-21G. Pancreatic beta cells exhibit key features of bona fidehuman pancreatic beta cells and are glucose responsive. FIGS. 21A-21D:Quantitative PCR analysis of selected gene transcripts in sorted GFP⁺pancreatic beta cells (green bars (center bar for each x-axis group in21A-21D)), GFP⁻ populations (blue bars (left bar for each x-axis groupin 21A-21D)) and human islet preparations (black bars (right bar foreach x-axis group in 21A-21D)). Results shown relative to the endogenouscontrol GAPDH. RQ=relative quantification. Values are average ±SD. n=4independent experiments for hESC-derived cell populations at days 19-20and n=3 for human islets. FIG. 21E: Insulin, human C-peptide, andproinsulin content relative to DNA in pancreatic beta cells at day 19.Data presented is average ±standard error (n=3 independent experiments,technical duplicates). FIG. 21F: Transmission electron microscopy imagesof pancreatic beta cells at day 20. One of three experiments withsimilar results is shown. Scale bar=500 nm. Insets represent secretoryvesicles akin to granules present in bona fide human pancreatic betacells. FIG. 21G: Glucose-stimulated insulin secretion (GSIS) of humanislets and pancreatic beta cells at days 19-20. ‘Y’ axis indicates ratioof insulin secreted in low glucose conditions to that secreted in highglucose conditions. Values are average ±standard deviation (SD) (n=3 forhuman islets and n=10 for pancreatic beta cells).

FIGS. 22A-22B. Efficient processing of insulin in hESC derivedpancreatic beta cells. A: Western blot analysis of proinsulin processingto insulin in pancreatic beta cells at indicated time points. FIG. 22Ahuman islet preparation is shown for comparison. Proceeding from left toright, Western blot panels of the following are provided: i) two humanislet preparations, ii) hES9.3 cells sampled at days 17, 19 and 21, iii)hES9.6 cells sampled at days 17, 19 and 21, and iv) hES9.10 cellssampled at days 17 and 19, are shown. The Western blot was cropped toconserve space, enabling tubulin levels to be shown (as a highermolecular weight protein, tubulin ran above proinsulin and insulin inthe gel subjected to Western blot). FIG. 22B: Quantification ofproinsulin processing in FIG. 19E and panel A. n=3 for each time pointof pancreatic beta cells and n=4 for human islets.

FIGS. 23A-23D. Pancreatic beta cells remain glucose responsive aftershort-term transplantation. FIG. 23A: Levels of circulating humanc-peptide measured in sera of mice 7-10 days after transplantation witheither 4000 human islets or 5.0×10⁶ direct-differentiated cells(containing approximately 1.15×10⁶ pancreatic beta cells). Fasting andchallenge sera were collected following an overnight fast and 1 hourafter intraperitoneal glucose challenge, respectively. Dashed lineseparates raw data from serum c-peptide measurements normalized to thenumber of pancreatic beta cells present in each human islet graft (4000human islets transplanted each containing about 1000 cells,approximately 50% of which are pancreatic beta cells, hence 2.0×10⁶pancreatic beta cells present in grafts total). n=5 for human islets andn=12 for hES-derived grafts. FIG. 23B: Hematoxylin and Eosin staining ofday 14 graft. k=kidney, g=graft. Representative data from one of threemice are shown. FIG. 23C: Immunofluorescence staining of differentiatedhES grafts 2 weeks post-transplantation for human C-peptide, Glucagon(GCG), Somatostatin (SST), PDX1, NKX6.1, and NKX2.2. Representative datafrom one of three mice are shown. FIG. 23D: Blood glucose (BG) levels ofmice treated with streptozotocin to ablate endogenous beta-cells (STZ)followed by transplantation (Tx) of pancreatic beta cell containingclusters either at day 0 or day 4, as indicated (n=8, two independentdifferentiation experiments). Values are average ±standard deviation.Statistical significance was calculated using two-tailed t-test.p=*<0.05, **<0.01, and ***<0.001. Control (CTRL)=6-9 animals.

FIG. 24. Schematic illustration of the method for directlydifferentiating pancreatic beta cells from embryonic stem cells. Themethod involves extended exposure of cells to epidermal growth factorand/or keratinocyte growth factor (EGF/KGF). Progenitor cells can bemaintained and expanded for extended periods of time. The method alsoinvolves optimized combinations and concentrations of known factorscapable of inducing endocrine differentiation, marked by NGN3expression, while maintaining the expression of PDX1/NKX6.1, resultingin the rapid and efficient generation of functional pancreatic betacells. The method further involves exposure of cells to two novelfactors, i.e., vitamin C and BayK-8644, which facilitate the generationof functional, improved pancreatic beta cells that are or resemble bonafide pancreatic beta cells.

FIGS. 25A-25I. Characterization of cells differentiated using thedisclosed method of direct differentiation. FIG. 25A: Live image ofdifferentiated cells expressing GFP under the control of the endogenousinsulin promoter. FIG. 25B: Flow-based analysis of hormones glucagon andinsulin (detected by human C-peptide staining) FIG. 25C: Histogram ofC-peptide staining shown in C highlighting higher expression levels.FIG. 25D: Immunofluorescence analysis of pancreatic markers humanc-peptide (C-PEP), Glucagon (GCG), Somatostatin (SST), Chromagranin A(CHGA), the transcription factors NKX6.1, PDX1, PAX6, NKX2.2, NEUROD1,and ISL1 on sections of d19-21 clusters generated using the disclosedmethod. FIG. 25E: Static Glucose Stimulated Insulin Secretion (sGSIS) atd19. FIG. 25F: Flow-based analysis of d20 differentiation shows up to75% c-peptide positive pancreatic beta cells. FIG. 25G: Transmissionelectron microscopy image of d19 cells exhibits prominent mature Insulinvesicles. FIG. 25H: Static Glucose Stimulated Insulin Secretion (sGSIS)of sorted and re-aggregated pancreatic beta cells. FIG. 25I: ELISAanalysis for human C-peptide in serum of mice transplanted with d19-20clusters after 1 week. Values shown are after an overnight fast and 30minutes post i.p. Glucose bolus.

FIG. 26. Schematic illustration of transplantation experimentshighlighting the importance of physiological oxygen tension (about 5%)during differentiation. (iv) Transplantation of pancreatic beta cellsgenerated under ambient oxygen conditions resulted in the loss ofcritical markers in many cells. Some pancreatic beta cells, however,maintain their phenotype and function immediately. (v) and (vi)Differentiation under physiological oxygen levels, either starting atprogenitor (v) or pluripotent stage (vi) results in the generation orproduction of pancreatic beta cells in vitro. These pancreatic betacells are more resilient to stress caused by transplantation and mostcells function immediately, representing a key advance over current celltherapy approaches.

FIGS. 27A-27D: Beta cells can be efficiently produced underphysiological oxygen levels and exhibit reduced death upon exposure tohypoxia. FIG. 27A: Differentiating cell clusters were transferred at day10 or 11 from an ambient to physiological oxygen atmosphere. FIG. 27B:Micrographs of clusters generated at physiological oxygen concentrationsat day 20 under light microscopy (left picture) or fluorescentmicroscopy showing prominent GFP expression (right picture). FIG. 27C:Representative flow-based analysis of hormones glucagon and insulin(detected by human C-peptide staining) at day 20 and quantification ofhuman C-peptide-positive cells of total cells and glucagon-expressingcells of C-peptide cells. n=3 independent experiments. FIG. 27D:Differentiated clusters containing beta cells generated at eitherambient or physiological oxygen levels were transferred to control (20%O₂) or hypoxic (1% O₂) culture conditions and dead cells were assayed atindicated time points.

DETAILED DESCRIPTION

Methods and compositions described herein provide populations ofpancreatic beta cells. Such cells are useful for treatment of diabetes.

The pancreatic beta cells can be generated using the methods andcompositions described herein. For example, by differentiation of lessdifferentiated cells, such as by differentiation of stem cells, inducedpluripotent stem cells, and progenitor cells that are of the endodermalof pancreatic lineage. In some cases, the pancreatic beta cells can begenerated from adult cells that have been directly converted into theendodermal or pancreatic lineage by procedures described herein (seealso PCT/US2014/048358, filed Jul. 25, 2014, which is herebyincorporated by reference in its entriety).

Pancreatic endodermal progenitor cells can then be differentiated intopancreatic beta cells by contact with a composition that includesheparin, zinc salts, a TGF-β inhibitor (e.g., Alk5 inhibitor), a BMP4signaling inhibitor (e.g., LDN-193189), a T3 thyroid hormone, cysteine,an anti-oxidant (e.g., vitamin E or a derivative thereof such asTrolox), an Axl kinase inhibitor (e.g., R428), a Notch SignalingInhibitor (e.g., Compound E), a Ca²⁺ channel agonist (e.g. BayK-8644),vitamin C, and combinations thereof to generate populations of partiallyor fully differentiated pancreatic beta-like cells. These cells can thenbe cultured to generate 3D aggregates that are useful fortransplantation to

To obtain definitive endoderm cells that can be differentiated intopancreatic endodermal progenitor cells, differentiated cells can bere-directed from an established lineage to the endoderm lineage. Thedefinitive endoderm cells generated as described herein express Sox17,Foxa2, Cerberus1, CXCR4, or a combination thereof. Compositions andprocedures for such preparation of definitive endoderm cells aredescribed herein. For example, embryonic or adult fibroblasts can beredirected into the endoderm lineage to form definitive endoderm cellsby inducing expression of pluripotency factors at the same time as theembryonic fibroblasts are mixed with a first composition containing aTGFβ family member such as Activin A and a WNT activator such asCHIR99021 and/or lithium chloride. The yield of definitive endodermcells can be increased by including growth factors (e.g., epidermalgrowth factor (EGF), basic fibroblast growth factor (bFGF), or acombination thereof), phospho-L-ascorbic acid, a histone deacetylaseinhibitor (e.g., Na butyrate), a histone demethylase inhibitor (e.g.,parnate), a DNA methyltransferase inhibitor (e.g., RG108), an adenosineagonist (e.g., 5′-N-ethylcarbox-amido-adenosine (NECA)), and/or a G9ahistone methyl-transferase inhibitor in the first composition (e.g., inthe culture medium).

Pancreatic progenitor cells can be obtained from definitive endodermcells by contacting or mixing the definitive endoderm cells with asecond composition. For example, pancreatic progenitor cells can beobtained from definitive endoderm cells by contacting or mixing thedefinitive endoderm cells with a second composition that includes a TGFβreceptor inhibitor, a hedgehog pathway inhibitor, a retinoic acidreceptor agonist, or a combination thereof. The yield of pancreaticprogenitor cells can be increased by including a WNT activator,2-phospho-L-ascorbic acid, a Notch signaling inhibitor, a BMP4 signalinginhibitor, growth factors (e.g., epidermal growth factor (EGF), basicfibroblast growth factor (bFGF), fibroblast growth factor 7 (FGF7),fibroblast growth factor 10 (FGF10), or a combination thereof).

An example of a process for obtaining definitive endoderm cells andpancreatic precursor cells is outlined in FIG. 4A and in Table 1, shownbelow.

TABLE 1 Stage* Stages I & II Stage III Stage IV Days 0-12 Days 12-16Days 16-25 Definitive Pancreatic Functional Endodermal Cells ProgenitorCells Pancreatic that Express that Express Beta-Like Cells Sox17 & Foxa2Pdx1 & Nkx6.1 that Express (Cer & Cxcr4) (Hnf6 & Pax6) glucagon &insulin Factor Add laminin, nicotinamide, B27, etc. (per Approach 1Approach 2 Schroeder 2006) TGFβ family 6 days 6 days member pluripotencyActivin A + factors, then pluripotency 6 days of factors, then Activin A6 days of Activin A WNT 6 days With activator pluripotency pluripotencyfactors, then factors 6 days of 6 days, then LiCl 6 days of LiCl RAR Addretinoic Agonist acid at day 12 for 1 day or when Sox17 & Foxa2expressed TGFβ Add A83-01 at receptor day 12 for 1 day, Inhibitor orwhen Sox17 & Foxa2 expressed; then add A83-01 for 3 more days Vitamin CAdd Add pVc at day Add pVc Phospho-L- 12 for 1 day, or when cellsascorbic acid when Sox17 & express trisodium salt Foxa2 Pdx1 & Nkx6.1(pVc) expressed; then day 0-12 add pVc for 3 more days Hedgehog AddLDE225 at Inhibitor day 12 for 1 day, or when Sox17 & Foxa2 expressed;then add LDE225 for 3 more days G9a histone Add Bix- methyl- 01294transferase day 0-6 Inhibitor p38 mitogen- Add SB203580 activated whencells protein express (MAP) kinase Pdx1 & Nkx6.1 inhibitor *Stage I isinduction of pluripotency, which can include contacting cells withActivin A and LiCl; but if the cells are already pluripotent (or of anappropriate stem cell type) then stage I may not be needed. See FIG. 1A.

In another example, a related procedure for obtaining definitiveendoderm cells and pancreatic precursor cells is outlined in Table 2,shown below.

TABLE 2 Generating Definitive Endodermal Generating Progenitor CellsGenerating Functional from fibroblasts Pancreatic Progenitor CellsBeta-Like Cells Stage Posterior Endodermal foregut-like EndodermalFunctional Conversion progenitor progenitor Beta-Like Initiation Days7-28 cells cells Cells Days 0-7 (cDE) (cPF) (cPE) (cPB) Factor Inducesexpression of SOX 7, Induces Induces FOXA2, expression of expression ofInduces HNF4α, FOXA2, INSULIN, PDX1, expression of HNF6, HNF6, SOX9,NKX6.1, SOX17, SOX9, PDX1 & NEUROD1, and FOXA2 and/or PDX1 NKX6.1.NKX2.2 Pluripotency 7 days factor recovery expression after inducingpluripotency factor expression Growth EGF & EGF & FGF7 & Factors bFGFbFGF FGF10 (2 days) TGFβ family Activin A member (e.g., (100 ng/ml)Activin A) WNT CHIR99021 CHIR99021 CHIR99021 activator (e.g., LiCl,CHIR-99021) RAR Retinoic Acid Agonist (2 days) TGFβ A83-01 A83-01 A83-01receptor (2 days) (3 days) Inhibitor Vitamin C Vitamin C HedgehogGDC-0449 Inhibitor Histone Na butyrate Na butyrate deacetylase inhibitorHistone Parnate Parnate demethylase inhibitor DNA methyl- RG108 RG108transferase inhibitor Adenosine 5′-N- 5′-N- agonist ethylcarbox-ethylcarbox- amido- amido- adenosine adenosine (NECA) (NECA) NotchCompound E Compound E Inhibitor (2 days) (3 days) BMP4 LDN-193189signaling (2 days) inhibitor Agonist of Extendin-4 Glucagon-like (3days) Peptide-1 polyADP- Nicotinamide ribose (3 days) synthetaseinhibitor Adenylyl Forskolin cyclase activator Gluco- Dexamethasonecorticoid receptor agonist Ca²⁺ channel BayK-8644 agonist

The exact type of factors employed and the incubation times can bevaried over what is exemplified in Tables 1 and 2, as is described inmore detail below.

Definitive Endoderm Induction

Definitive endoderm cells can be identified by their expression ofSox17, Foxa2, Cerberus1, CXCR4, or a combination thereof.

Starting Cells

Definitive endoderm cells can be obtained in several ways. The extent ofdifferentiation of the initial cell type selected can impact whatinitial steps are chosen to make definitive endoderm cells. For example,if pluripotent stem cells are used as the starting cells, induction ofpluripotency factors is not needed. However, if differentiated cells areemployed as the starting cells, then introduction and/or induction ofpluripotency factor expression facilitates conversion of the cells intodefinitive endoderm cells. There is no need to induce full pluripotencyin the starting cells. Instead, the starting cells can be converteddirectly to definitive endodermal cells.

A variety of cell types can therefore be used to generate definitiveendodermal cells. For example, definitive endodermal cells can begenerated from induced pluripotent stem cells (iPSCs), from embryonicstem cells, from multipotent stem cells, and by manipulation of othercell types. In some instances, definitive endodermal cells can begenerated by re-directing cells from one lineage to another. Forexample, the endodermal cells can be obtained from starting cells ofepidermal lineage, hematopoietic lineage, endothelial lineage, musclecell lineage, epithelial cell lineage, and/or neural cell lineage.Examples of starting cells that can be converted into endodermal cellsinclude fibroblasts, epidermal cells, lymphocytes, hepatocytes,myoblasts, neurons, osteoblasts, osteoclasts, T-cells, and combinationsthereof. As illustrated by experiments described herein, embryonicfibroblasts can be redirected to an endodermal lineage by the methodsdisclosed herein.

Starting cells for generation of definitive endoderm cells can include aselected cell population that contains nonpluripotent cells that areinduced to transiently express one or more pluripotency factors such asSSEA1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2,E-cadherin, UTF-1, Oct4,Klf4, Sox2, c-Myc, a short hairpin RNA (shRNA)against p53, or a combination thereof. The selected population caninclude pluripotent stem cells, induced pluripotent stem cells (iPSCs),embryonic stem cells, multipotent stem cells, and combinations thereof.The selected cell population can be re-directed from one lineage to theendodermal lineage. For example, partially and/or fully differentiatedcells of the epithelial cell lineage, hematopoietic lineage, endothelialcell lineage, muscle cell lineage, neural cell lineage, and combinationsthereof can be re-directed to the endodermal lineage and to becomepancreatic progenitor cells.

The initial starting population of cells can be a nonpluripotent cellpopulation, for example, a cell population that does not expressdetectable levels of endogenous NANOG, but that is induced totransiently express pluripotency factors while being directed to theendodermal lineage.

Expression of endogenous or recombinantly introduced pluripotencyfactors can also be induced to facilitate redirection of a selected cellpopulation to the endodermal lineage. For example, pluripotentexpression vectors can be transfected into a selected cell population,and expression of the pluripotency factors encoded by those expressionvectors can be induced. The pluripotent expression vectors can beintegrated into the genomes of the cells, or the pluripotent expressionvectors can be maintained episomally for the time needed to redirect thecells to the endodermal lineage. Episomal introduction and expression ofpluripotency factors is desirable because the mammalian cell genome isnot altered by insertion of the episomal vectors and because theepisomal vectors are lost over time. Hence, use of episomal expressionvectors allows expression of pluripotency factors for the short timethat is needed to convert nonpluripotent mammalian cells to definitiveendodermal progenitor cells, while avoiding possible chromosomalmutation and expression of pluripotency factors during later stages ofdifferentiation into pancreatic progenitor cells and pancreatic betacells.

Episomal plasmid vectors encoding p53 suppression factors and otherpluripotency factors can be introduced into mammalian cells as describedfor example, in Yu et al., Human induced pluripotent stem cells free ofvector and transgene sequences, Science 324(5928): 797-801 (2009);United States Patent Application 20120076762, and Okita et al., A moreefficient method to generate integration free human iPS cells, NATUREMETHODS 8: 409-412 (2011), the contents of which are specificallyincorporated herein by reference in their entireties.

For example, the pluripotency factors can be encoded within andexpressed from an episomal vector that has EBNA-1 (Epstein-Barr nuclearantigen-1) and oriP, or Large T and SV40ori sequences so that thevectors can be episomally present and replicated without incorporationinto a chromosome.

Cells from various lineages can be induced to a stem cell-like phenotypeby procedures described by United States Patent Application Nos.20130059385, 20120190059, 20110110899, 20100267141, 20100233804 andWO/2011/123572, the contents of which are specifically incorporatedherein by reference in their entireties.

The pluripotency factors can be introduced into mammalian cells in theform of DNA, protein or mature mRNA by a technique such as lipofection,binding with a cell membrane-permeable peptide, liposomaltransfer/fusion, or microinjection. When in the form of DNA, a vectorsuch as a virus, a plasmid, or an artificial chromosome can be employed.Examples of viral vectors include retrovirus vectors, lentivirus vectors(e.g., according to Takahashi, K. and Yamanaka, S., Cell, 126: 663-676(2006); Takahashi, K. et al., Cell, 131: 861-872 (2007); Yu, J. et al.,Science, 318: 1917-1920 (2007)), adenovirus vectors (e.g., Okita K, etal., Science 322: 949 (2008)), adeno-associated virus vectors, andSendai virus vectors (Proc Jpn Acad Ser B Phys Biol Sci. 85: 348-62,2009), the contents of each of which references are incorporated hereinby reference in their entireties. Also, examples of artificialchromosome vectors that can be used include human artificial chromosome(HAC), yeast artificial chromosome (YAC), and bacterial artificialchromosome (BAC and PAC) vectors. As a plasmid, a plasmid for mammaliancells can be used (e.g., Okita K, et al., Science 322: 949 (2008)). Avector can contain regulatory sequences such as a promoter, an enhancer,a ribosome binding sequence, a terminator, and a polyadenylation site,so that a pluripotency factor can be expressed. A vector may furthercontain, if desired, a selection marker sequence such as a drugresistant gene (e.g., a neomycin resistant gene, an ampicillin resistantgene, and a puromycin resistant gene), a thymidine kinase gene, and adiphtheria toxin gene, a reporter gene sequence such as a greenfluorescent protein (GFP), β glucuronidase (GUS), FLAG, or combinationsthereof. Also, the above vector may have LoxP sequences located beforeand after the segment encoding the pluripotency factor to permitcleavage at the ends of the pluripotency factor segment (before andafter) or at both ends of the segment encoding a promoter and thepluripotency factor after introduction into the mammalian cells.

The nucleic acid segment encoding a pluripotency factor can be operablylinked to a promoter. The promoter is typically selected from promoterswhich are functional in mammalian cells, although prokaryotic promotersand promoters functional in other eukaryotic cells may be used. Thepromoter can be derived from promoter sequences of viral or eukaryoticgenes. For example, it may be a promoter derived from the genome of acell in which expression is to occur. However, a heterologous promoteris often desirable. Examples of eukaryotic promoters that can beemployed include those promoters that function in a ubiquitous manner(such as promoters of a-actin, b-actin, tubulin) or, alternatively, atissue-specific manner (such as promoters of the genes for pyruvatekinase). Tissue-specific promoters can be specific for lymphocytes,dendritic cells, skin, brain cells and epithelial cells. Examples ofpromoters include CD2, CD11c, keratin 14, Wnt-1 and Rhodopsin promoters.An epithelial cell promoter such as SPC can be used. Viral promoters mayalso be used, for example the Moloney murine leukemia virus longterminal repeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTRpromoter or the human cytomegalovirus (CMV) IE promoter. The promotersemployed for expression of pluripotency factors can be induciblepromoters that respond to specific stimuli. An inducible promoter is apromoter that is capable of directly or indirectly activatingtranscription of one or more DNA sequences or genes in response to aninducer. In the absence of an inducer, the DNA sequences or genes willnot be transcribed. The inducer can be a chemical agent such as aprotein, metabolite, growth regulator, phenolic compound, steroid, or aphysiological stress imposed directly by, for example heat, orindirectly through the action of a pathogen or disease agent such as avirus. It may be advantageous for the promoters to be inducible so thatthe levels of expression of the heterologous gene can be regulatedduring the life-time of the cell. In addition, any of these promotersmay be modified by the addition of further regulatory sequences, forexample enhancer sequences. Chimeric promoters may also be usedcomprising sequence elements from two or more different promotersdescribed above.

There is no need to induce pluripotency to the extent that pluripotencymarkers such as Nanog and Rex1 are expressed. Instead, cells are exposedto or induced to express pluripotency factors to redirect the cells fromtheir current differentiation lineage and toward the endodermaldifferentiation lineage.

The selected cell population is treated with Activin A and lithiumchloride to generate definitive endoderm cells. Addition ofphospho-L-ascorbic acid and/or a G9a histone methyl-transferaseinhibitor can increase the yield of definitive endoderm cells from theselected cell population. Such treatment is for a time and at aconcentration of Activin A, lithium chloride, phospho-L-ascorbic acidand/or a G9a histone methyl-transferase inhibitor that is sufficient toinduce differentiation or re-direction of the selected cell populationto the endoderm lineage. Further details on treatment of a selected cellpopulation to generate definitive endoderm cells are provided below.

Growth Factors

The composition used to convert starting cells to definitive endodermcells can contain growth factors such as epidermal growth factor, basicfibroblast growth factor, fibroblast growth factor (e.g., FGF7 and/orFGF10), and combinations thereof. Such growth factors can also improveconversion of definitive endoderm cells to pancreatic progenitor cells.

Epidermal growth factor can stimulate cell growth, cell proliferation,and cellular differentiation Human epidermal growth factor is a smallprotein (approximately 6045 daltons) with about 53 amino acids and threeintramolecular disulfide bonds. Epidermal growth factor is availablecommercially, for example, from MP Biomedicals (see, e.g., mpbio.com),PeproTech (see, e.g., peprotech.com), and Cell Signaling Technology(see, e.g., cellsignal.com).

The fibroblast growth factor (FGF) family is comprised of at least ninepolypeptides that show a variety of biological activities toward cellsof mesenchymal, neuronal and epithelial origin. All FGFs have twoconserved cysteine residues and share 30-50% sequence identity at theamino acid level.

Basic fibroblast growth factor can help maintain cells in anundifferentiated state. Basic fibroblast growth factor is commerciallyavailable, for example, from BD Biosciences (see, e.g.,bdbiosciences.com), and EMD Millipore (see, e.g., Millipore.com).

Fibroblast growth factor 7 (FGF-7) is also called keratinocyte growthfactor (KGF) and is encoded by the FGF7 gene. KGF/FGF-7 was originallyisolated from the conditioned medium of a human embryonic lungfibroblast cell line as a mitogen that is specific for epithelial cells.The transcript for KGF/FGF-7 can be detected in stromal but notepithelial cells from various epithelial tissues. It has been proposedthat KGF is a mesenchymal cell-derived paracrine growth factor thatspecifically stimulates epithelial cell growth. The KGF cDNA encodes a194 amino acid precursor protein from which the N-terminal 31 amino acidresidues are cleaved to generate the mature KGF. Human KGF exhibitsspecies cross-reactivity and is active on mouse, monkey, and porcinecells. A high affinity receptor for KGF has been cloned and shown to bean alternatively spliced isoform of FGF R2/bek. Whereas FGF R2 binds FGFacidic and FGF basic but not KGF with high affinity, the alternatelyspliced KGF/FGF-7 R can bind KGF and FGF acidic with high affinity. FGF7is commercially available from R&D Systems (see website atrndsystems.com/Products/251-KG).

Fibroblast growth factor 10 (FGF10) is a protein that in humans isencoded by the FGF10 gene. The human FGF10 cDNA encodes a 208 amino acidresidue protein with a hydrophobic amino-terminal signal peptide. Basedon its in vitro biological activities and in vivo expression pattern,FGF10 has been proposed to play unique roles in the brain, in lungdevelopment, wound healing and limb bud formation. FGF10 is commerciallyavailable from R&D Systems (see website atrndsystems.com/product_results.aspx?m=1448 orrndsystems.com/Products/345-FG).

As illustrated herein epidermal growth factor, basic fibroblast growthfactor, fibroblast growth factor 7, fibroblast growth factor 10, andcombinations thereof can facilitate reprogramming of differentiatedcells to the endodermal lineage. Experiments described herein show thataddition of such growth factors to a selected population of cells duringor after expression of pluripotency factors can increase the proportionand yield of cells that express endodermal markers. In particular,addition of such growth factors to starting cells induces those cells toexpress markers indicative of a endoderm phenotype such as Sox17, Foxa2,Cerberus 1 (Cer), C—X—C chemokine receptor type 4 (Cxcr4), or acombination thereof.

Similarly, epidermal growth factor, basic fibroblast growth factor,fibroblast growth factor 7, fibroblast growth factor 10, andcombinations thereof can facilitate reprogramming of definitiveendodermal cells to pancreatic progenitor cells. Experiments describedherein show that addition of such growth factors to a definitiveendodermal cells can increase the proportion and yield of cells thatexpress pancreatic progenitor cell markers. In particular, addition ofsuch growth factors to definitive endodermal cells induces those cellsto express markers indicative of the pancreatic progenitor phenotypesuch as Pdx1, Nkx6.1, Pax6, Hnf6, or a combination thereof.

In some cases, a combination of two growth factors is employed in thefirst or second composition, such as epidermal growth factor and basicfibroblast growth factor (or alternatively, for example, FGF7 andFGF10). In other cases, a combination of three growth factors, or acombination of four growth factors is employed.

For example, treatment of a selected population of cells with epidermalgrowth factor, basic fibroblast growth factor, fibroblast growth factor7, fibroblast growth factor 10, or combinations thereof can convert orinduce at least about 0.5%, or at least about 1%, or at least about 2%,or at least about 3%, or at least about 4%, or at least about 5%, or atleast about 6%, or at least about 7%, or at least about 8% of cells inthe selected mammalian cell population to express Sox17, Foxa2, Cerberus1 (Cer), C—X—C chemokine receptor type 4 (Cxcr4), or a combinationthereof. Similarly, treatment of a definitive endodermal population ofcells with epidermal growth factor, basic fibroblast growth factor,fibroblast growth factor 7, fibroblast growth factor 10, or combinationsthereof can convert or induce at least about 0.5%, or at least about 1%,or at least about 2%, or at least about 3%, or at least about 4%, or atleast about 5%, or at least about 6%, or at least about 7%, or at leastabout 8% of cells in the selected mammalian cell population to expressPdx1, Nkx6.1, Pax6, Hnf6, or a combination thereof.

For example, concentrations of such growth factors that are at leastabout 1 ng/ml, or at least about 2 ng/ml, or at least about 3 ng/ml, orat least about 5 ng/ml in a first composition are useful for convertingstarting cells to definitive endodermal cells. Similarly, concentrationsof such growth factors of at least about 1 ng/ml, or at least about 2ng/ml, or at least about 3 ng/ml, or at least about 5 ng/ml, or about 10ng/ml in a second composition are useful for converting definitiveendodermal cells to pancreatic progenitor cells.

To increase the proportion of cells that express markers indicative ofan endoderm phenotype or pancreatic progenitor phenotype, a selectedpopulation of cells is contacted or mixed with one or more growthfactors for a time and at a concentration sufficient to differentiate orre-direct the cells to an endoderm lineage.

The time of contacting or mixing one or more growth factors with astarting population of cells to generate definitive endodermal cells canvary, for example, from about 2 days to about 20 days, or from 3 days toabout 15 days, or from 4 days to about 10 days, or from 5 days to about9 days, or from 6 days to about 8 days, or about 7 days.

The time of contacting or mixing one or more growth factors with adefinitive endodermal population of cells to generate pancreaticprogenitor cells can vary, for example, from about 3 days to about 130days, or from 5 days to about 120 days, or from 7 days to about 110days, or from 10 days to about 100 days, or from 20 days to about 95days, or about 30 days to about 95 days.

The growth factor(s) can be added to a selected cell population duringinduced pluripotency and while directing the cells into the endodermlineage.

The growth factors can be employed in the compositions and methodsdescribed herein in a variety of amounts and/or concentrations. Forexample, the growth factor(s) can be employed at a concentration ofabout 0.01 ng/ml to about 1 mg/ml, or about 0.1 ng/ml to about 300 ng/mlin a solution, or about 0.5 ng/ml to about 100 ng/ml in a solution, orabout 1 ng/ml to about 50 ng/ml, or about 5 ng/ml to about 20 ng/ml in asolution, or about 10 ng/ml in a solution. In a dry formulation, theepidermal growth factor and basic fibroblast growth factor can bepresent in amounts of about 0.01 mg to about 1000 mg, or about 0.1 mg toabout 100 mg, or about 1 mg to about 10 mg.

TGFβ Family Members

Transforming growth factor beta (TGF-β) is a protein that is involved inthe control of proliferation, cellular differentiation, and otherfunctions in most cells. It is a cytokine that has a role in immunity,cancer, bronchial asthma, heart disease, diabetes, Hereditaryhemorrhagic telangiectasia, Marfan syndrome, Vascular Ehlers-Danlossyndrome, Loeys-Dietz syndrome, Parkinson's disease and AIDS. Proteinsof the TGF-beta family are active as homodimers or heterodimers, withthe two chains being linked by a single disulfide bond. Examples ofTGF-β family members include the Activin/Inhibin subfamily, thedecapentaplegic-Vg-related (DVR) related subfamily (that includes bonemorphogenetic proteins and the growth differentiation factors), and theTGF-β subfamily.

Activin A is a member of the TGFβ family first identified in late 1980sas an inducer of follicle-stimulating hormone. Activin A is highlyconserved in evolution and throughout the animal kingdom. It regulates avariety of biologic processes including cell proliferation,hematopoiesis, wound healing, and fibrosis. Activin A signals throughthe activin type I (Alk2, 4, or 7) and type II (ActRII or ActRIIB)receptors and shares with TGFβ the activation of the Smad cascade. See,Phillips et al., Cytokine Growth Factor Rev. 20(2): 153-64 (2009);Werner, Cytokine Growth Factor Rev. 17(3): 157-71 (2006).

As shown herein, addition of Activin A to a selected population of cellsduring expression of pluripotency factors increases the proportion andyield of definitive endodermal like cells generated. In particular,addition of Activin A to cells that express SSEA1, SSEA-3, SSEA-4,TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, ora combination thereof, increases the proportion of cells that expressmarkers indicative of a definitive endoderm phenotype such as Sox17,Foxa2, Cerberus 1 (Cer), C—X—C chemokine receptor type 4 (Cxcr4), or acombination thereof. For example, after treatment of a selectedpopulation of cells with Activin A, at least about 5%, or at least about10%, or at least about 15%, or at least about 20%, or at least about22%, or at least about 23%, or at least about 24%, or at least about 25%of cells in the selected mammalian cell population express Sox17, Foxa2,Cerberus 1 (Cer), C—X—C chemokine receptor type 4 (Cxcr4), or acombination thereof.

To increase the proportion of cells that express markers indicative of adefinitive endoderm phenotype, a selected population of cells iscontacted or mixed with Activin A for a time and at a concentrationsufficient to differentiate or re-direct the cells to an endodermallineage.

The time of contacting or mixing Activin A with the selected populationof cells can vary, for example, from about 1 days to about 20 days, orfrom 2 days to about 18 days, or from 3 days to about 16 days, or from 4days to about 15 days, or from 5 days to about 14 days, or from about 6days to about 12 days.

Activin A can be added to a selected cell population during inducedpluripotency and while directing the cells into the definitive endodermlineage.

Activin A can be used at a variety of concentrations, for example, atabout 5 ng/ml to about 200 ng/ml, or from about 10 ng/ml to about 175ng/ml, or from about 15 ng/ml to about 150 ng/ml, or from about 20 ng/mlto about 150 ng/ml, or from about 25 ng/ml to about 125 ng/ml, or fromabout 30 ng/ml to about 100 ng/ml, or from about 35 ng/ml to about 80ng/ml, or from about 40 ng/ml to about 60 ng/ml, or about 50 ng/ml.

Activin A is available commercially from various suppliers, for example,from Invitrogen, PeproTech, StemRD, R&D Systems, and other vendors.

Nucleic acid and protein sequences for Activin A are available, forexample, in the sequence database maintained by the National Center forBiotechnology Information. One example of a human Activin A amino acidsequence is available as accession number EAW94141.1 (GI:119614547) andprovided below as SEQ ID NO:1.

  1 MPLLWLRGFL LASCWIIVRS SPTPGSEGHS AAPDCPSCAL  41AALPKDVPNS QPEMVEAVKK HILNMLHLKK RPDVTQPVPK  61AALLNAIRKL HVGKVGENGY VEIEDDIGRR AEMNELMEQT 121SEIITFAESG TARKTLHFEI SKEGSDLSVV ERAEVWLFLK 161VPKANRTRTK VTIRLFQQQK HPQGSLDTGE EAEEVGLKGE 201RSELLLSEKV VDARKSTWHV FPVSSSIQRL LDQGKSSLDV 241RIACEQCQES GASLVLLGKK KKKEEEGEGK KKGGGEGGAG 281ADEEKEQSHR PFLMLQARQS EDHPHRRRRR GLECDGKVNI 321CCKKQFFVSF KDIGWNDWII APSGYHANYC EGECPSHIAG 361TSGSSLSFHS TVINHYRMRG HSPFANLKSC CVPTKLRPMS 401MLYYDDGQNI IKKDIQNMIV EECGCS

WNT Activators

The WNT signaling pathway includes a series of events that occur when aWNT protein binds to a cell-surface receptor of a Frizzled receptorfamily member. Such events result in the activation of Dishevelledfamily proteins which inhibit a complex of proteins that includes axin,GSK-3, and the protein APC to degrade intracellular beta-catenin. Theresulting enriched nuclear beta-catenin enhances transcription byTCF/LEF family transcription factors. A WNT activator can thereforeinclude an agent that activates TCF/LEF-mediated transcription in acell. WNT activators can be selected from true WNT agonists that bindand activate a Frizzled receptor family member including any and all ofthe WNT family proteins, an inhibitor of intracellular beta-catenindegradation, activators of TCF/LEF, and inhibitors of GSK-3.

As illustrated herein WNT activators are useful for converting startingcells to definitive endodermal cells and for converting definitiveendodermal cells to pancreatic progenitor cells.

Examples of WNT activators that can be employed include one or more ofthe following compounds:

-   -   CHIR99021        (6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinonitrile);    -   1-azakenpaullone        (9-Bromo-7,12-dihydro-pyrido[3′,2′:2,3]azepino[4,5-b]indol-6(5H)-one),        BIO ((2′Z,3′E)-6-Bromoindirubin-3′-oxime);    -   AR-A014418        (N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea);    -   Indirubin-3′-monoxime;    -   5-Iodo-indirubin-3′-monoxime;    -   kenpaullone        (9-Bromo-7,12-dihydroindolo-[3,2-d][1]benzazepin-6(5H)-one);    -   SB-415286        (3-[(3-Chloro-4-hydroxyphenyl)amino]-4-(2-nitro-phenyl)-1H-pyrrole-2,5-dione);    -   SB-216763        (3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione);    -   Maybridge SEW00923SC (2-anilino-5-phenyl-1,3,4-oxadiazole);    -   (Z)-5-(2,3-Memylenedioxyphenyl)imidazolidine-2,4-dione,    -   TWS119        (3-(6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yloxy)phenol);    -   CHIR98014        (N2-(2-(4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)pyrimidin-2-ylamino)ethyl)-5-nitropyridine-2,6-diamine);    -   SB415286        (3-(3-chloro-4-hydroxyphenylamino)-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione);    -   Tideglusib (also known as NP031112, or NP-12;        1,2,4-Thiadiazolidine-3,5-dione,        2-(1-naphthalenyl)-4-(phenylmethyl));    -   LY2090314 (1H-Pyrrole-2,5-dione,        3-imidazo[1,2-a]pyridin-3-yl-4-[1,2,3,4-tetrahydro-2-(1-piperidinylcarbonyl)pyrrolo[3,2,1-jk][1,4]benzodiazepin-7-yl]);    -   lithium salt (e.g., LiCl); or    -   any combination thereof.

WNT activators can also include small-interfering RNAs (siRNA, CellSignaling) that act as GSK-inhibitors, lithium (Sigma), kenpaullone(Biomol International, Leost, Metal (2000) Eur J Biochem 267,5983-5994), 6-Bromoindirubin-30-acetoxime (Meyer, L et al (2003) ChemBiol 10, 1255-1266), SB 216763 and SB 415286 (Sigma-Aldrich), andFRAT-family members and FRAT-derived peptides that prevent interactionof GSK-3 with axin. An overview is provided by Meijer et al, (2004)Trends in Pharmacological Sciences 25, 471-480, which is herebyincorporated by reference in its entirety. WNT activators (GSK3inhibitors) that can be used in the compositions and methods describedherein can also include those disclosed in US 20120329152 by Pera etal., which is specifically incorporated herein in its entirety.

The WNT activators can, for example, be CHIR99021, SB216763, TWS119,CHIR98014, Tideglusib, SB415286, LY2090314, or any combination thereof.In some embodiments, the WNT activators can be CHIR99021, whosestructure is shown below.

The WNT activators can also be in the form of a salt or hydrate of anyof the foregoing compounds.

To increase the proportion of cells that express markers indicative ofan endodermal or pancreatic progenitor phenotype, a selected populationof cells is contacted or mixed with one or more WNT activators for atime and at a concentration sufficient to differentiate or re-direct thecells to an endodermal and/or pancreatic progenitor lineage.

The time of contacting or mixing WNT activator(s) with a startingpopulation of cells (to generate definitive endodermal cells) can vary,for example, from about 2 days to about 50 days, or from 3 days to about40 days, or from 4 days to about 35 days, or from 5 days to about 33days, or from 6 days to about 30 days, or about 14 to 28 days. The timeof contacting or mixing WNT activator(s) with a population of definitiveendodermal cells (to generate pancreatic progenitor cells) can vary, forexample, from about 3 days to about 130 days, or from 5 days to about120 days, or from 7 days to about 110 days, or from 10 days to about 100days, or from 20 days to about 95 days, or about 30 days to about 95days.

WNT activators can be added to a selected starting cell populationduring induced pluripotency and while directing the cells into theendoderm lineage. WNT activators can also be added to a definitiveendodermal cell population to be converted to pancreatic progenitorcells.

The WNT activators can be employed in the compositions and methodsdescribed herein in a variety of amounts and/or concentrations. Forexample, the WNT activators can be employed at a concentration of about0.01 micromolar to about 1 millimolar in a solution, or about 0.1micromolar to about 100 micromolar in a solution, or about 0.5micromolar to about 10 micromolar in a solution, or about 1 micromolarto about 5 micromolar in a solution. In a dry formulation, the WNTactivators can be present in amounts of about 0.01 mg to about 1000 mg,or about 1 mg to about 100 mg, or about 1 mg to about 10 mg can bepresent in amounts of about 0.01 mg to about 1000 mg, or about 0.1 mg toabout 100 mg, or about 1 mg to about 10 mg.

Methods and assays for determining a level of WNT activation or GSK-3inhibition are available to a skilled person and include, for example,the methods and assays described in Liao et al., Endocrinology, 145(6):2941-2949 (2004); and in U.S. Pat. No. 8,323,919, both of which arespecifically incorporated by reference herein in their entireties.

Lithium

Lithium is an activator of WNT. The lithium can be in the form of asalt, where the anion includes, but is not limited to, chloride,bromide, carbonate, citrate, sulfate, or other biologically compatiblemonovalent anion (see, for example, US 2004/0028656 and WO 2008/055224).

As shown herein, addition of lithium salts to a selected population ofcells during expression of pluripotency factors increases the proportionand yield of definitive endodermal like cells generated. In particular,addition of lithium salts to cells that express SSEA1, SSEA-3, SSEA-4,TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, ora combination thereof, increases the proportion of cells that expressmarkers indicative of a definitive endoderm phenotype such as Sox17,Foxa2, Cerberus 1 (Cer), C—X—C chemokine receptor type 4 (Cxcr4), or acombination thereof. For example, after treatment of a selectedpopulation of cells with lithium salts at least about 10%, or at leastabout 15%, or at least about 20%, or at least about 22%, or at leastabout 23%, or at least about 24%, or at least about 25% of cells in theselected mammalian cell population express Sox17, Foxa2, Cerberus 1(Cer), C—X—C chemokine receptor type 4 (Cxcr4), or a combinationthereof.

To increase the proportion of cells that express markers indicative of adefinitive endoderm phenotype, a selected population of cells iscontacted or mixed with lithium salts for a time and at a concentrationsufficient to differentiate or re-direct the cells to an endodermlineage.

The time of contacting or mixing lithium salts with the selectedpopulation of cells can vary, for example, from about 1 days to about 20days, or from 2 days to about 18 days, or from 3 days to about 16 days,or from 4 days to about 15 days, or from 5 days to about 14 days, orfrom about 6 days to about 12 days.

Lithium salts can be added to a selected cell population during inducedpluripotency and while directing the cells into the definitive endodermlineage. Lithium salts can be employed at a variety of concentrations,for example, at about 0.01 mM to about 10 mM, or from about 0.05 mM toabout 9 mM, or from about 0.1 mM to about 8 mM, or from about 0.2 mM toabout 7 mM, or from about 0.3 mM to about 6 mM, or from about 0.4 mM toabout 5 mM, or from about 0.5 mM to about 3 mM, or about 1 mM.

Vitamin C (Ascorbic Acid and/or Phospho-L-Ascorbic Acid)

As shown herein, addition of vitamin C or phospho-L-ascorbic acid to aselected population of cells during expression of pluripotency factorsincreases the proportion and yield of definitive endodermal like cellsgenerated. In particular, addition of phospho-L-ascorbic acid to cellsthat express SSEA1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E,ALP, Sox2, E-cadherin, UTF-1, Oct4, or a combination thereof, increasesthe proportion of cells that express markers indicative of a definitiveendoderm phenotype such as Sox17, Foxa2, Cerberus 1 (Cer), C—X—Cchemokine receptor type 4 (Cxcr4), or a combination thereof. Forexample, after treatment of a selected population of cells with vitaminC or phospho-L-ascorbic acid at least about 10%, or at least about 15%,or at least about 20%, or at least about 22%, or at least about 23%, orat least about 24%, or at least about 25% of cells in the selectedmammalian cell population express Sox17, Foxa2, Cerberus 1 (Cer), C—X—Cchemokine receptor type 4 (Cxcr4), or a combination thereof.

Also as shown herein, addition of 2-phospho-L-ascorbic to definitiveendoderm cells increases the proportion and yield of Pdx1⁺ and/orNkx6.1⁺ pancreatic progenitor cells. For example, more than about 5%, ormore than about 10%, or more than about 15%, or more than about 20%, orabout 25% of the cells were Pdx1 positive and/or Nkx6.1 positive. The2-phospho-L-ascorbic acid can therefore be added to cells that alreadyexpress Sox17, Foxa2, Cerberus 1 (Cer), C—X—C chemokine receptor type 4(Cxcr4), or a combination thereof.

In addition, the presence of vitamin C or phospho-L-ascorbic acid incell culture media can improve conversion of pancreatic progenitor cellsto pancreatic beta cells.

To increase the proportion of cells that express markers indicative of adefinitive endoderm phenotype, a pancreatic progenitor phenotype, or apancreatic beta cell phenotypes a selected starting population of cellsis contacted or mixed with vitamin C or phospho-L-ascorbic acid for atime and at a concentration sufficient to differentiate or convert thecells to the desired phenotype.

The time of contacting or mixing vitamin C or phospho-L-ascorbic acidwith the selected population of cells can vary, for example, from about1 days to about 50 days, or from 2 days to about 45 days, or from 3 daysto about 40 days, or from 4 days to about 35 days, or from 5 days toabout 33 days, or from about 6 days to about 30 days, or for about 20-25days.

Phospho-L-ascorbic acid or vitamin C can be added to a starting cellpopulation during induced pluripotency and while directing the cellsinto the definitive endoderm lineage.

Phospho-L-ascorbic acid or vitamin C can be employed at a variety ofconcentrations, for example, at about 1 μM to about 1000 μM, or fromabout 10 μM to about 700 μM, or from about 20 μM to about 500 μM, orfrom about 30 μM to about 400 μM, or from about 40 μM to about 350 μM,or from about 45 μM to about 300 μM, or from about 50 μM to about 310μM, or about 280 μM.

To increase the proportion of cells that express markers indicative of apancreatic progenitor cell phenotype, a selected population ofdefinitive endoderm cells is contacted or mixed with2-phospho-L-ascorbic acid or vitamin C for a time and at a concentrationsufficient to differentiate at least some of the cells into pancreaticprogenitor cells.

The time of contacting or mixing phospho-L-ascorbic acid or vitamin Cwith the definitive endoderm cells or pancreatic progenitor cells canvary, for example, from about 1 days to about 50 days, or from 2 days toabout 45 days, or from 3 days to about 40 days, or from 4 days to about35 days, or from 5 days to about 33 days, or from about 6 days to about30 days, or for about 20-25 days.

Phospho-L-ascorbic acid and/or vitamin C can be added to definitiveendoderm cells while directing those cells to differentiate intopancreatic progenitor cells. Phospho-L-ascorbic acid or vitamin C can beemployed at a variety of concentrations, for example, at about 1 μM toabout 1000 μM, or from about 20 μM to about 700 μM, or from about 50 μMto about 500 μM, or from about 100 μM to about 400 μM, or from about 150μM to about 350 μM, or from about 200 μM to about 325 μM, or from about250 μM to about 310 μM, or about 280 μM.

Phospho-L-ascorbic acid and/or vitamin C can be added to pancreaticprogenitor cells while directing those cells to differentiate intopancreatic beta cells. Phospho-L-ascorbic acid or vitamin C can beemployed at a variety of concentrations, for example, at about 1 μM toabout 100 μM, or from about 10 μM to about 80 μM, or from about 25 μM toabout 75 μM, or from about 30 μM to about 70 μM, or from about 35 μM toabout 65 μM, or from about 40 μM to about 60 μM, or from about 45 μM toabout 55 μM, or about 50 μM.

G9a Histone Methyl-Transferase Inhibitor

The G9a histone methyltransferase enzymatically methylates histonelysines. The G9a histone methyltransferase is also named euchromatinhistone methyltransferase 2 (EHMT2). Histone lysine methylation hasimportant roles in the organization of chromatin domains and theregulation of gene expression.

As shown herein, addition of inhibitors of G9a histone methyltransferaseto a selected population of cells during expression of pluripotencyfactors increases the proportion and yield of definitive endodermal likecells generated. In particular, addition of such inhibitors to cellsthat express SSEA1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E,ALP, Sox2, E-cadherin, UTF-1, Oct4, or a combination thereof increasesthe proportion of cells that express markers indicative of a definitiveendoderm phenotype such as Sox17, Foxa2, Cerberus 1 (Cer), C—X—Cchemokine receptor type 4 (Cxcr4), or a combination thereof. Forexample, after treatment of a selected population of cells withinhibitors of G9a histone methyltransferase at least about 10%, or atleast about 15%, or at least about 20%, or at least about 22%, or atleast about 23%, or at least about 24%, or at least about 25% of cellsin the selected mammalian cell population express Sox17, Foxa2, Cerberus1 (Cer), C—X—C chemokine receptor type 4 (Cxcr4), or a combinationthereof.

To increase the proportion of cells that express markers indicative of adefinitive endoderm phenotype, a selected population of cells iscontacted or mixed with an inhibitor of G9a histone methyltransferasefor a time and at a concentration sufficient to differentiate orredirect the cells to an endoderm lineage.

The time of contacting or mixing a G9a histone methyltransferaseinhibitor with the selected population of cells can vary, for example,from about 1 days to about 15 days, or from 1 days to about 14 days, orfrom 2 days to about 13 days, or from 3 days to about 10 days, or from 3days to about 9 days, or from about 4 days to about 8 days, or fromabout 5 days to about 7 days.

The G9a histone methyltransferase inhibitors can be added duringinduction of pluripotency, for example, for 2 days, or for 3 days, orfor 4 days, or for 5 days, or for about 6 days while the cells areexposed to, or are induced to express, pluripotency factors.

G9a histone methyltransferase inhibitors can be used at a variety ofconcentrations, for example, at about 0.01 μM to about 10 μM, or fromabout 0.05 μM to about 9 μM, or from about 0.1 μM to about 8 μM, or fromabout 0.2 μM to about 7 μM, or from about 0.3 μM to about 6 μM, or fromabout 0.4 μM to about 5 μM, or from about 0.5 μM to about 3 μM, or about1 μM.

A variety of G9a histone methyl-transferase inhibitors can be employed.For example, the G9a histone methyl-transferase inhibitor can beBix-01294, chaetocin, 3-deazaneplanocin hydrochloride, UNC 0224, UNC0638, UNC 0646, and combinations thereof. G9a histone methyl-transferaseinhibitors can be obtained commercially, for example, from TocrisBioscience (see website at tocris.com/pharmacologicalBrowser.php?ItemId=236264&Type=Inhibitors#.UdYcHeDD85s). Another type of G9ahistone methyl-transferase inhibitor is BRD4770 (Yuan et al., ACS Chem.Biol. 7(7): 1152-1157 (2012)(incorporated herein by reference in itsentirety).

Histone Deacetylase (HDAC) Inhibitors

Histone deacetylases (HDAC) are a class of enzymes that remove acetylgroups from an ε-N-acetyl lysine amino acid on a histone. ExemplaryHDACs include those Class I HDAC: HDAC1, HDAC2, HDAC3, HDAC8; and ClassII HDACs: HDAC4, HDAC5, HDAC6, HDAC7A, HDAC9, HDAC10. Type I mammalianHDACs include: HDAC1, HDAC2, HDAC3, HDAC8, and HDAC11. Type II mammalianHDACs include: HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC1.

As illustrated herein use of one or more histone deacetylase inhibitorscan facilitate conversion of starting cells into the endodermal lineage.The histone deacetylase inhibitors can inhibit one or more of thesehistone deacetylases. In some instances the histone deacetylaseinhibitors are inhibitors of HDAC1.

Inhibitors of HDACs (HDAC inhibitors) can include, for example,butyrate, small molecular weight carboxylates (e.g., less than about 250amu), hydroxamic acids, benzamides, epoxyketones, cyclic peptides, andhybrid molecules. (See, for example, Drummond et al., Annu Rev PharmacolToxicol 45: 495-528 (2005), (including specific examples therein) whichis hereby incorporated by reference in its entirety). Non-limitingexamples of negative regulators of type I/II HDACs include:

-   -   Sodium butyrate, phenyl butyrate, or butyrate;    -   Suberoylanilide Hydroxamic Acid (SAHA; also called Vorinostat        and MK0683), which inhibits the activities of HDAC1 and HDAC3,        for example, with IC50 values of about 10 nM and 20 nM,        respectively;    -   BML-210 (N1-(2-aminophenyl)-N8-phenyl-octanediamide, available        from Sigma-Aldrich); in HeLa extracts, the IC50 of BML-210 for        inhibition of HDAC activity can, for example, be about 80 μM;    -   Depudecin (e.g., (−)-Depudecin;        4,5:8,9-Dianhydro-1,2,6,7,11-pentadeoxy-D-threo-D-ido-undeca-1,6-dienitol),        which can, for example, have an IC50 for HDAC1 of about 4.7 μM;    -   HC Toxin        ((6R,9S,14aR)-3,6R-dimethyl-9S-(7-((S)-oxiran-2-yl)-7-oxoheptyl)decahydropyrrolo[1,2-a][1,4,7,10]tetraazacyclododecine-1,4,7,        10-tetranone, available from Cayman Chemical); HC Toxin is a        cell-permeable, reversible inhibitor of histone deacetylases        (HDACs) (e.g., IC₅₀=30 nM);    -   Scriptaid        (N-Hydroxy-1,3-dioxo-1H-benz[de]isoquinoline-2(3H)-hexananmide);    -   Phenylbutyrate (e.g., sodium phenylbutyrate), Sodium Butyrate,        pivaloyloxymethyl butyrate (Pivanex, AN-9);    -   Valproic Acid ((VPA) and other short chain fatty acids),    -   Suramin (e.g., Suramin Sodium);    -   Trichostatin A (TSA;        (R,2E,4E)-6-(4-(dimethylamino)benzoyl)-N-hydroxy-4-methylhepta-2,4-dienamide),        for example, with an IC50 of about 1.8 nM;    -   APHA Compound 8        (3-(1-Methyl-4-phenylacetyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamide),        which is HDAC class I-selective;    -   Apicidin        (Cyclo[(2S)-2-Amino-8-oxodecanoyl-1-methoxy-L-tryptophyl-L-isoleucyl-(2R)-2-piperidine-carbonyl]),        which is a potent histone deacetylase with, for example, an        IC50=0.7 nM;    -   Trapoxin B        (3,6-dibenzyl-9-[6-(oxiran-2-yl)-6-oxohexyl]-1,4,7,10-tetrazabicyclo[10.3.0]pentadecane-2,5,8,11-tetrone),        an HDAC1 inhibitor with, for example, an IC50 of about 0.1 nM;    -   Chlamydocin        ((3R)-3-benzyl-6,6-dimethyl-9-[6-[(2R)-oxiran-2-yl]-6-oxohexyl]-1,4,7,10-tetrazabicyclo[10.3.0]pentadecane-2,5,8,11-tetrone),        with, for example, an IC50 of about 0.15 nM;    -   Depsipeptide (also known as romidepsin, FR901228 or FK228;        (1S,4S,7Z,10S,16E,21R)-7-ethylidene-4,21-di(propan-2-yl)-2-oxa-12,13-dithia-5,8,20,23-tetrazabicyclo[8.7.6]tricos-16-ene-3,6,9,19,22-pentone);    -   CI-994 (also known as acetyldinaline or Tacedinaline;        4-acetamido-N-(2-aminophenyl)benzamide), with, for example, a Ki        of 0.05 for HDAC1;    -   MS-27-275 (also known as MS275 or entinostat;        pyridin-3-ylmethyl-N-[[(4-[(2-aminophenyl)carbamoyl]phenyl]methyl]carbamate),        with, for example, an IC50 of about 0.1-1 μM;    -   MGCD0103 (also known as Mocetinostat,        N-(2-aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl]benzamide),        with, for example, an IC50 of about 0.1 μM;    -   NVP-LAQ-824 (also known as Dacinostat or LAQ824,        (E)-N-hydroxy-3-[4-[[2-hydroxyethyl-[2-(1H-indol-3-yl)ethyl]amino]methyl]phenyl]prop-2-enamide),        with, for example, an IC50 for HDAC1 of about 0.003-0.008 μM;    -   CBHA (also known as m-carboxycinnaminic acid bishydroxamic acid;        N-hydroxy-3-[(E)-3-(hydroxyamino)-3-oxoprop-1-enyl]benzamide);    -   JNJ16241199 (also known as R306465;        N-hydroxy-2-(4-(naphthalen-2-ylsulfonyl)piperazin-1-yl)pyrimidine-5-carboxamide),        a potent inhibitor of HDAC1 with, for example, IC50 values of        about 30 to 300 nM;    -   Tubacin (also known as 537049-40-4, AClO7Y2P, CHEMBL356769,        CTK8E6516, DIOX-H_003551, Y6280;        N-[4-[(2R,4R,6S)-4-[(4,5-diphenyl-1,3-oxazol-2-yl)sulfanylmethyl]-6-[4-(hydroxymethyl)phenyl]-1,3-dioxan-2-yl]phenyl]-N′-hydroxyoctanediamide),        with, for example, a Ki for HDAC1 of about 0.028 μM;    -   A-161906 (7-[4-(4-cyanophenyl)phenoxy]-heptanohydroxamic acid);    -   Proxamide (see WO2007031853A2);    -   Oxamflatin        ((E)-5-[3-(benzenesulfonamido)phenyl]-N-hydroxypent-2-en-4-ynamide);    -   3C1-UCHA (6-(3-chlorophenylureido)caproic hydroxamic acid);    -   AOE (2-amino-8-oxo-9,10-epoxydecanoic acid);    -   CHAP31        ((2S)—N′-hydroxy-N-[(2R)-3-(4-methoxyphenyl)-1-[[(2S,3R)-3-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]-2-(pyrrolidine-2-carbonylamino)octanediamide);        or    -   any combination thereof.        See WO2007031853A2, which is incorporated by reference herein in        its entirety, for structures of many of these HDAC inhibitors.

Other inhibitors include, for example, dominant negative forms of theHDACs (e.g., catalytically inactive forms), siRNA inhibitors of theHDACs, and antibodies that specifically bind to the HDACs. Inhibitorsare available, e.g., from BIOMOL International, Fukasawa, MerckBiosciences, Novartis, Gloucester Pharmaceuticals, Aton Pharma, TitanPharmaceuticals, Schering AG, Pharmion, MethylGene, and Sigma Aldrich.

In some embodiments the HDAC inhibitor(s) can include sodium butyrate.

The time of contacting or mixing HDAC inhibitor(s) with the selectedpopulation of cells can vary, for example, from about 2 days to about 50days, or from 3 days to about 40 days, or from 4 days to about 35 days,or from 5 days to about 33 days, or from 6 days to about 30 days, orabout 21-28 days.

The HDAC inhibitor(s) can be added to a selected cell population duringinduced pluripotency and while directing the cells into the endodermlineage.

The HDAC inhibitor can be employed in the compositions and methodsdescribed herein in a variety of amounts and/or concentrations. Forexample, the HDAC inhibitor can be employed at a concentration of about1 micromolar to about 20 millimolar, or about 10 micromolar to about 15millimolar, or about 25 micromolar to about 5 millimolar, or about 40micromolar to about 1 millimolar, or about 60 micromolar to about 0.5millimolar, or about 0.1 millimolar in a solution. In a dry formulation,the HDAC inhibitor can be present in amounts of about 0.01 mg to about100 mg, or about 0.05 mg to about 50 mg, or about 0.1 mg to about 25 mg,or about 1 mg to about 8 mg. For example, entinostat (MS275) has beenadministered during clinical trials at dosages of about 4-5 mg/m² (Piliet al., Br J Cancer 106(1): 77-84 (2012)), where mg/m² is mg per bodysurface area of patient. The adult average body surface is about 2.2 m²and formulae are available converting height and weight into bodysurface area.

Histone Demethylase Inhibitors

Histone demethylases remove methyl groups from histone. Thelysine-specific demethylase 1 (LSD1, also called KDM1, AOF2, or BHC110)is a histone demethylase that suppresses gene expression by convertingdi-methylated lysines on histone H3 to monomethylated and unmethylatedlysines. Histone methylation can influence epigenetic patterns of geneexpression due to association with active promoters. As illustratedherein use of one or more inhibitors of histone demethylase enzymes canfacilitate conversion of differentiated cells into the endodermallineage.

Exemplary inhibitors of histone demethylase include, but are not limitedto, parnate (also called tranylcypromine sulfate) or an equivalent saltof parnate, and phenelzine (Nardil, 2-phenylethylhydrazine). See, also,Huang et al., Proc Natl Acad Sci USA. 104(19): 8023-8028 (2007); Bi, X.et al., Bioorg. Med. Chem. Lett. 16:3229-3232 (2006); InternationalPatent Application Nos. WO2007/021839 and WO2008/127734. MAO inhibitorscan also serve as epigenetic modulators.

In some embodiments, the histone demethylase inhibitor is parnate

The time of contacting or mixing histone demethylase inhibitor(s) withthe selected population of cells can vary, for example, from about 2days to about 50 days, or from 3 days to about 40 days, or from 4 daysto about 35 days, or from 5 days to about 33 days, or from 6 days toabout 30 days, or about 21-28 days.

The histone demethylase inhibitor(s) can be added to a selected cellpopulation during induced pluripotency and while directing the cellsinto the endoderm lineage.

The histone demethylase inhibitor can be employed in the compositionsand methods described herein in a variety of amounts and/orconcentrations. For example, the histone demethylase inhibitor can beemployed at a concentration of about 0.01 micromolar to about 20micromolar, or about 0.05 micromolar to about 10 micromolar, or about0.1 micromolar to about 5 micromolar, or about 0.5 micromolar to about 3micromolar, or about 1 micromolar to about 3 micromolar, or about 1micromolar in a solution. In a dry formulation, the lysine-specificdemethylase 1 inhibitor can be present in amounts of about 0.01 mg toabout 100 mg, or about 0.05 mg to about 50 mg, or about 0.1 mg to about25 mg, or about 1 mg to about 8 mg.

DNA Methyltransferase (DNMT) Inhibitors

DNA methyltransferases are enzymes that transfer methyl groups to DNA.Inhibitors of DNA methyltransferases can reactivate the expression ofgenes that have been repressed by DNA methylation. As illustratedherein, DNA methyltransferase inhibitors can improve the conversion ofstarting cells to definitive endodermal cells.

Exemplary DNA methyltransferase (DNMT) inhibitors can include antibodiesthat bind to DNA methyltransferases, dominant negative variants of DNAmethyltransferases, and siRNA and antisense nucleic acids that suppressexpression of DNMT. DNA methyltransferase inhibitors include, but arenot limited to, RG108 (available, e.g., from Sigma-Aldrich), 5-aza-C(5-azacitidine or azacitidine) (see, e.g., Schermelleh, et al., NatureMethods 2:751-6 (2005)), 5-aza-T-deoxycytidine (5-aza-CdR) (see, e.g.,Zhu, Clinical Medicinal Chemistry 3(3):187-199 (2003)), decitabine (see,e.g., Gore, Nature Clinical Practice Oncology 2:S30-S35 (2005)),doxorubicin (see, e.g., Levenson, Molecular Pharmacology 71:635-637(2007)), EGCG ((−)-epigallocatechin-3-gallate) (see, e.g., Fang, et al.,Cancer Research 63:7563-7570 (2003)), RG108 (see, e.g., Carninci, etal., WO2008/126932, incorporated herein by reference) and zebularine(see, Carninci, supra).

In some embodiments, the DNA methyltransferase inhibitor is RG108, whichhas the following structure.

The time of contacting or mixing DNA methyltransferase inhibitor(s) withthe selected population of cells can vary, for example, from about 2days to about 50 days, or from 3 days to about 40 days, or from 4 daysto about 35 days, or from 5 days to about 33 days, or from 6 days toabout 30 days, or about 7-28 days.

The DNA methyltransferase inhibitor(s) can be added to a selectedstarting cell population during induced pluripotency and while directingthe cells into the endoderm lineage. The DNA methyltransferase inhibitorcan be employed in the compositions and methods described herein in avariety of amounts and/or concentrations. For example, the DNAmethyltransferase inhibitor can be employed at a concentration of about0.01 micromolar to about 20 micromolar, or about 0.03 micromolar toabout 10 micromolar, or about 0.05 micromolar to about 5 micromolar, orabout 0.1 micromolar to about 2 micromolar, or about 0.2 micromolar toabout 1 micromolar, or about 0.5 micromolar in a solution. In a dryformulation, the DNA methyltransferase inhibitor can be present inamounts of about 0.01 mg to about 100 mg, or about 0.05 mg to about 50mg, or about 0.1 mg to about 25 mg, or about 1 mg to about 8 mg.

Adenosine Receptor Agonists

Adenosine receptor agonists bind to and/or activate adenosine receptors.Adenosine receptor agonists can activate any, two or more, or all of theadenosine receptor subtypes, including A₁, A_(2a), A_(2b), and A₃adenosine receptors. Various adenosine receptor subtypes and agonistsare described in the scientific literature, including, e.g., Muller C E,“Medicinal chemistry of adenosine A3 receptor ligands,” Curr Top MedChem. 3(4):445-62, 2003; Cristalli G et al., “Medicinal chemistry ofadenosine A2A receptor agonists,” Curr Top Med Chem. 3(4):387-401, 2003;Gao Z G, et al., “Partial agonists for A(3) adenosine receptors,” CurrTop Med Chem. 4(8):855-62, 2004; Zablocki J A et al., “Partial A(1)adenosine receptor agonists from a molecular perspective and theirpotential use as chronic ventricular rate control agents during atrialfibrillation (AF),” Curr Top Med Chem. 4(8):839-54, 2004; Dalpiaz A etal., “Adenosine A(1) receptor: analysis of the potential therapeuticeffects obtained by its activation in the central nervous system,” CurrMed Chem. 9(21):1923-37, 2002 November; Cristalli G et al., “Medicinalchemistry of adenosine A2A receptor agonists,” Curr Top Med Chem.3(4):387-401, 2003; Gao Z G et al., “Allosteric modulation of theadenosine family of receptors,” Mini Rev Med Chem. 5(6):545-53, 2005June; Headrick J P et al., “A3 adenosine receptor-mediated protection ofthe ischemic heart,” Vascul Pharmacol. 42(5-6):271-9, 2005 April-May,Epub 2005 Apr. 19; Hutchinson S A et al., “A(1) adenosine receptoragonists: medicinal chemistry and therapeutic potential,” Curr PharmDes. 10(17):2021-39, 2004; Cerqueira M D, “The future of pharmacologicstress: selective A2A adenosine receptor agonists,” Am J Cardiol.94(2A):33D-40D, 2004 Jul. 22, discussion 40D-42D; Lukashev D et al.,“Targeting hypoxia—A(2A) adenosine receptor-mediated mechanisms oftissue protection,” Drug Discov Today. 9(9):403-9, 2004 May 1; Yan L etal., “Adenosine receptor agonists: from basic medicinal chemistry toclinical development, “Expert Opin Emerg Drugs. 8(2):537-76, 2003November; Sullivan G W, “Adenosine A2A receptor agonists asanti-inflammatory agents,” Curr Opin Investig Drugs. 4(11):1313-9, 2003November; Jacobson K A et al., “Adenosine receptors as therapeutictargets, Nat Rev Drug Discov. 5(3):247-64, 2006 March; Gross, G. J. andAuchampach, J. A. “Reperfusion injury: Does it exist?” J. Mol. Cell.Cardiol. (2006), 42: 12; Baraldi, P. G. et al., “Ligands for A2Badenosine receptor subtype” Curr. Med. Chem. (2006) 13:3467; Yuzlenko,O.; Kiec-Kononowicz, K. “Potent adenosine A1 and A2A receptorsantagonists: Recent developments.” Curr. Med. Chem. (2006), 13:3609,Cronstein, B. N. Adenosine receptors and wound healing, revised,” TheScientific World Journal (2006) 6:984; Akkari, R. et al., “Recentprogress in the development of Adenosine receptor ligandsanti-inflammatory drugs,” Curr. Top. Med. Chem. (2006) 6:1375; Vallon,V. et al., “Adenosine and kidney function,” Physiol. Rev. (2006) 86:901;Bours, M. J. L., et al., “Adenosine 5′-triphosphate and adenosine asendogenous signaling molecules in immunity and inflammation,” Pharmacol.Ther. (2006) 112:358, the contents of which are specificallyincorporated herein by reference in their entireties.

A variety of adenosine receptor agonists can be used in the methods andcompositions described herein. For example, as illustrated herein5′-N-ethylcarbox-amido-adenosine (NECA) can improve the conversion ofstarting cells to definitive endodermal cells. The structure of NECA isshown below.

Other adenosine receptor agonists that can be employed include thefollowing.

The 8-butylamino-adenosine compound is a partial agonist for theadenosine A₁ receptor. Prototypical A₂ agonists include2-[p-(2-carboxyethyl)phenethyl-amino]-5′-N-ethylcarboxamidoadenosine(CGS-2 1680) and HENECA. Another example of a NECA-modified adenosineanalog is4-(3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl)-cyclohexanecarboxylicacid methyl ester) from Adenosine Therapeutics. Agonists selective forthe A₁ adenosine receptor subtype include, but are not limited to,N⁶-cyclopentyladenosine (CPA), 2-chloro-N⁶-cyclopentyl-adenosine (CCPA),(2S)—N6-[2-endo-Norbornyl]adenosine ((S)-ENBA),N-(2-aminoethyl)-2-[4-[[2-[4-[[9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]purin-6-yl]amino]phenyl]acetyl]amino]phenyl]acetamidehydrate (ADAC), AMP579, NNC-21-0136, GR79236, CVT-510 (Tecadenoson), SDZWAG 994, and Selodenoson.

Agonists selective for the A_(2A) adenosine receptor subtype include,but are not limited to, NECA, CGS21680, DPMA, Binodenoson, ATL-146e, andCV-3146. Agonists selective for the A_(2B) adenosine receptor subtypeinclude, but are not limited to, LUF5835.

Agonists selective for the A₃ adenosine receptor subtype include, butare not limited to, IB-MECA, Cl-IB-MECA, LJ568, CP-608039, MRS3558, andMRS1898. In various embodiments, the agonist is AmP579, a mixedadenosine agonist with both A₁ and A₂ effects (Rhone-Poulenc Rorer,Collegeville, Pa.). The analog may also be DPMA, a selective adenosineA₂ receptor agonist, CPA, a selective adenosine A₁ receptor agonist,Benzyl-NECA, a selective A₃ receptor agonist, 2-chloroadenosine, anon-selective A₂/A₁ agonist, or NECA, a non-selective A₂/A₁ agonist.

The time of contacting or mixing adenosine receptor agonists with theselected population of cells can vary, for example, from about 2 days toabout 50 days, or from 3 days to about 40 days, or from 4 days to about35 days, or from 5 days to about 33 days, or from 6 days to about 30days, or from about 7 to about 28 days, or from about 14 to about 21days.

The adenosine receptor agonists can be added to a selected starting cellpopulation during induced pluripotency and while directing the cellsinto the endoderm lineage. The adenosine receptor agonists can beemployed in the compositions and methods described herein in a varietyof amounts and/or concentrations. For example, the adenosine receptoragonists can be employed at a concentration of about 0.01 micromolar toabout 20 micromolar, or about 0.03 micromolar to about 10 micromolar, orabout 0.05 micromolar to about 5 micromolar, or about 0.1 micromolar toabout 2 micromolar, or about 0.2 micromolar to about 1 micromolar, orabout 0.5 micromolar in a solution. In a dry formulation, the adenosinereceptor agonists can be present in amounts of about 0.01 mg to about100 mg, or about 0.05 mg to about 50 mg, or about 0.1 mg to about 25 mg,or about 1 mg to about 8 mg.

Expansion of Endodermal Cells

Endodermal cells can be expanded by culturing the cells in a mediumcontaining an activator of WNT signaling and an inhibitor of TGFβsignaling. The WNT activator and the TGFβ signaling inhibitor can be anyof those listed above. One example of a useful WNT activator forexpansion is CHIR99021. An example of a TGFβ signaling inhibitor usefulfor cellular expansion is A83-01. Addition of two growth factors (e.g.,EGF and bFGF) can improve the expansion and serial passage of thedefinitive endodermal cells. The cells proliferate rapidly with anaverage doubling time of 2 days. After 15 passages an increase in cellnumber was obtained of at least a trillion-fold.

The expanded cells maintained their epithelial colony morphology and hada posterior foregut-like phenotype as determined by immunofluorescencestaining for SOX17, FOXA2, HNF4α, HNF6, and SOX9. Followinggastrulation, definitive endoderm migrates to form the primitive gut,which initially consists of a single flattened sheet of cells surroundedby mesoderm. Simultaneously, as migration is occurring, the primitivegut is becoming regionalized along the dorsal-ventral andanterior-posterior axes into foregut, midgut, and hindgut domains. Theforegut region gives rise to many of the major organ systems such as theliver, thyroid, lungs, the upper airways, the biliary system, stomach,and pancreas. Hence, a posterior foregut-like phenotype is indicative ofcells that can give rise to pancreatic progenitor cells.

When the methods and compositions described herein are employed, highlevels of expression of multiple posterior foregut progenitor genetranscripts are detected in expanded posterior foregut-like cells,including SOX17, FOXA2, HNF1A, HNF1B, HNF4A, HNF6, SOX9 and PDX1, ascompared to parental fibroblasts. In contrast, ectodermal marker geneSOX1, mesodermal marker gene BRACHYURY, and pluripotency marker genesOCT4 and NANOG were not induced. Notably, the episomal vectors wereundetectable by qPCR assays in established posterior foregut-like cells(abbreviated as cPF cells).

Transplantation of expanded cPF cells under the kidney capsule of immunedeficient mice did not result in any tumor formation even afterprolonged periods of up to 12 weeks in vivo. Analysis of the cPF graftsdemonstrated epithelial structures expressing differentendoderm-specific markers, including E-cadherin, HNF4α, PDX1, SOX9, andpan-cytokeratin. Thus, the cPF cells can be greatly expanded in culturewhile maintaining their posterior foregut endodermal phenotype.

The cPF cells can be differentiated into more committed pancreaticendodermal progenitor cells using the methods described below.

For example, the cPF cells can first be cultured in the presence ofgrowth factors, an inhibitor of TGFβ, an inhibitor of Notch signaling, aretinoic acid receptor agonist, a hedgehog antagonist, an inhibitor ofBMP4 signaling, and combinations thereof for several days. The cPF cellscan be cultured in the presence of these agents for about 1 to 10 days,or about 2 to 5 days, or about 2 to 4 days. The growth factors employedcan be FGF7 and FGF10. The inhibitor of TGFβ can be A83-01. Theinhibitor of Notch signaling can be Compound-E. The hedgehog antagonistcan be GDC-0449, and the inhibitor of BMP4 signaling can be LDN-193189.

In a second step, the differentiating cells can be cultured for severalmore days, for example, in the presence of different growth factors(e.g., EGF), an agonist of glucagon-like peptide-1 (e.g., Extendin-4),an inhibitor of TGFβ (e.g., A83-01), an inhibitor of BMP4 signaling(e.g., LDN-193189), an activator of protein kinase C (e.g., phorbol12,13-dibutyrate), an inhibitor of Notch signaling (e.g., Compound-E),an inhibitor of polyADP-ribose synthetase (e.g. nicotinamide), andcombinations thereof. For example, the differentiating cells can becultured in the presence of these agents for about 1 to 10 days, orabout 2 to 5 days, or about 2 to 4 days.

The resulting pancreatic progenitor cells express FOXA2, HNF6, SOX9, andPDX1, NKX6.1, or combinations thereof.

Further details on generating pancreatic progenitor cells are describedbelow.

Pancreatic Progenitor Cells

Pancreatic progenitor cells can be identified by their expression ofPdx1, Nkx6.1, Pax6, Hnf6, FoxA2, Sox9, or a combination thereof.Pancreatic progenitor cells can be obtained from the definitive endodermcells and/or posterior foregut-like cells generated as described herein.The conversion to pancreatic progenitor cells can involve contacting ormixing the cells with a second composition. For example, pancreaticprogenitor cells can be obtained from definitive endoderm cells bycontacting or mixing the definitive endoderm cells with a secondcomposition that includes a TGFβ receptor inhibitor, a hedgehog pathwayinhibitor, a retinoic acid receptor agonist, or a combination thereof.The yield of pancreatic progenitor cells can be increased by including aWNT activator, 2-phospho-L-ascorbic acid, a Notch signaling inhibitor, aBMP4 signaling inhibitor, an activator of protein kinase C, growthfactors (e.g., epidermal growth factor (EGF), basic fibroblast growthfactor (bFGF), fibroblast growth factor 7 (FGF7), fibroblast growthfactor 10 (FGF10), or a combination thereof).

Starting Cells for Generating Pancreatic Progenitor Cells

Pancreatic progenitor cells can be obtained from definitive endodermcells, for example, cells that express Sox17, Foxa2, Cerberus 1 (Cer),C—X—C chemokine receptor type 4 (Cxcr4), or a combination thereof.Pancreatic progenitor cells can also be obtained from posteriorforegut-like cells, which express Sox17, Foxa2, Hnf4a, Sox9, or acombination thereof.

The starting cells are treated with a TGFβ receptor inhibitor, ahedgehog pathway inhibitor, a retinoic acid receptor agonist,2-phospho-L-ascorbic acid, an inhibitor of Notch signaling, an inhibitorof BMP4 signaling, one or more growth factors, or a combination thereof,to generate pancreatic progenitor cells. Additional factors that canimprove conversion to pancreatic progenitor cells include an agonist ofglucagon-like peptide-1, an inhibitor of BMP4 signaling, an activator ofprotein kinase C, an inhibitor of polyADP-ribose synthetase, andcombinations thereof. Several of these factors and details for use ofthese factors are described above in the section describing conversionof starting cells to definitive endodermal cells. Other factors and moredetails for converting definitive endoderm cells to pancreaticprogenitor cells are described below.

Retinoic Acid Receptor Agonists

As shown herein, addition of retinoic acid receptor agonists (withoutaddition of any growth factors) to definitive endoderm cells increasesthe proportion and yield of Pdx1⁺ and/or Nkx6.1⁺ pancreatic progenitorcells. For example, more than about 5%, or more than about 10%, or morethan about 15%, or more than about 20%, or about 25% of the cells werePdx1 positive and/or Nkx6.1 positive.

The retinoic acid receptor agonists can be added to cells that expressSox17, Foxa2, Cerberus 1 (Cer), C—X—C chemokine receptor type 4 (Cxcr4),or a combination thereof.

To increase the proportion of cells that express markers indicative of apancreatic progenitor cell phenotype, a selected population of cells(e.g., definitive endoderm cells) is contacted or mixed with a retinoicacid receptor agonist for a time and at a concentration sufficient todifferentiate at least some of the cells into pancreatic progenitorcells.

The time of contacting or mixing a retinoic acid receptor agonist withthe selected population of cells can vary, for example, from about 0.5days to about 12 days, or from 1 days to about 10 days, or from 1.5 daysto about 8 days, or from 2 days to about 7 days, or from 2.5 days toabout 6 days, or from about 3 days to about 5 days, or about 4 days.

Retinoic acid receptor agonists can be used at a variety ofconcentrations, for example, at about 0.01 μM to about 10 μM, or fromabout 0.05 μM to about 9 μM, or from about 0.1 μM to about 8 μM, or fromabout 0.3 μM to about 7 μM, or from about 0.5 μM to about 6 μM, or fromabout 0.75 μM to about 5 μM, or from about 1 μM to about 3 μM, or about2 μM.

A variety of retinoic acid receptor agonists can be employed. Examplesof retinoic acid receptor agonists include, for example, retinoic acid.The retinoic acid receptor agonists can be a naturally-occurringretinoid, or chemically synthesized retinoid, a retinoic acid receptoragonist compound free of retinoid skeleton, or a naturally-occurringsubstance having a retinoic acid receptor agonist activity. Examples ofthe natural retinoid having a RAR agonist activity include retinoic acid(stereoisomers of all-trans retinoic acid (all-trans RA) and9-cis-retinoic acid (9-cis RA) are known). A chemically synthesizedretinoid is known in this field (see, e.g., U.S. Pat. Nos. 5,234,926,and 4,326,055). Examples of the retinoic acid receptor agonist compoundfree of retinoid skeleton include Am80, AM580, TTNPB and AC55649.Examples of a naturally-occurring substance having a retinoic acidreceptor agonist activity includes honokiol and magnolol (Annual Reportof Research Institute for Biological Function 9:55-61, 2009). The RARagonist to be used in this step is preferably retinoic acid, AM580(4-[[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]carboxamide]benzoicacid), TTNPB(4-[[E]-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl]-1-propenyl]benzoicacid), AC55649 (4′-octyl-[1,1′-biphenyl]-4-carboxylic acid), morepreferably retinoic acid. While the concentration of an RAR agonist inthe medium is appropriately determined according to the kind of the RARagonist to be used, the concentration of retinoic acid when used as aRAR agonist is generally 0.1-100 μM, preferably 0.5-10 μM. Theconcentration of TTNPB when used as an RAR agonist is generally 0.02-20μM, preferably 0.05-10 μM. The concentration of AM580 when used as anRAR agonist is generally 0.02-20 μM, preferably 0.05-10 μM. Theconcentration of AC55649 when used as an RAR agonist is generally0.02-20 μM, preferably 0.1-10 μM.

TGFβ Receptor Inhibitors

TGFβ receptor is a serine/threonine kinase receptor. The transforminggrowth factor beta (TGFβ) signaling pathway is involved in many cellularprocesses in both the adult organism and in the developing embryoincluding cell growth, cell differentiation, apoptosis, cellularhomeostasis and other cellular functions. In spite of the wide range ofcellular processes that the TGFβ signaling pathway regulates, theprocess is relatively simple. TGFβ superfamily ligands bind to a type IIreceptor, which recruits and phosphorylates a type I receptor. The typeI receptor then phosphorylates receptor-regulated SMADs (R-SMADs) whichcan now bind the coSMAD SMAD4. R-SMAD/coSMAD complexes accumulate in thenucleus where they act as transcription factors and participate in theregulation of target gene expression. The TGFβ receptor inhibitor alsoincludes TGFβ receptor antagonists.

As shown herein, addition of inhibitors of TGFβ receptors (withoutaddition of any growth factors) to definitive endoderm cells increasesthe proportion and yield of Pdx1 and/or Nkx6.1⁺ pancreatic progenitorcells. For example, more than about 5%, or more than about 10%, or morethan about 15%, or more than about 20%, or about 25% of the cells werePdx1 positive and/or Nkx6.1 positive.

Also as shown herein, addition of inhibitors of TGFβ receptors (withoutaddition of any growth factors) to pancreatic progenitor cells canimprove the conversion of such cells to pancreatic beta cells. Forexample, more than about 0.1%, or more than about 0.2%, or more thanabout 0.5%, or more than about 1%, or about 2% of the cells, or morethan about 5%, or more than about 7% of cells treated with suchinhibitors of TGFβ receptors were positive for C-peptide and/or insulinproduction.

The TGFβ receptor inhibitors can be added to cells that express Sox17,Foxa2, Cerberus 1 (Cer), C—X—C chemokine receptor type 4 (Cxcr4), or acombination thereof. The TGFβ receptor inhibitors can be added to cellsthat express Pdx1 and/or Nkx6.1.

To increase the proportion of cells that express markers indicative of apancreatic progenitor cell phenotype or a mature pancreatic beta cellphenotype, a selected population of cells (e.g., definitive endodermcells or pancreatic progenitor cells) is contacted or mixed with aninhibitor of TGFβ receptors for a time and at a concentration sufficientto differentiate at least some of the cells into pancreatic progenitorcells, or pancreatic beta cells, respectively.

Examples of TGF-β inhibitors include, but are not limited to:

-   -   3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide        (also known as A83-01, and available from Tocris Bioscience),        which is a TGFβ kinase/Activin receptor like kinase (ALK5)        inhibitor that blocks the phosphorylation of Smad2 and inhibits        TGFβ-induced epithelial-to-mesenchymal transition;    -   SB431542 (also known as        4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide;        available from Tocris Bioscience), which is a potent and        selective inhibitor of the transforming growth factor-β (TGF-β)        type I receptor Activin receptor-like kinase ALK5 (IC₅₀=94 nM),        and its relatives ALK4 and ALK7;    -   4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide        (also known as SB 431542 and available from Tocris Bioscience; a        potent and selective inhibitor of TGF-β type I receptor Activin        receptor-like kinase ALK5 (e.g., with IC₅₀=94 nM), and its        relatives ALK4 and ALK7);    -   2-(3-(6-methylpyridine-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine        (ALK5 inhibitor or ALK5 inhibitor II) is a selective and        ATP-competitive inhibitor of the TGF-β family type I receptor        activin receptor-like kinase (ALK5). The ALK5 Inhibitor works by        preventing autophosphorylation of ALK5. It has also been shown        that ALK5 can replace Sox2 when reprogramming cells to induced        pluripotent stem (iPS) cells. Through inhibition of the TGF-β        pathway, ALK5 works to reprogram cells that have been transduced        with Oct4, Klf4, and c-M. It is a selective and ATP-competitive        inhibitor of TGF-β RI kinase (IC₅₀=23 nM, 4 nM and 18 nM for        binding, auto-phosphorylation and cellular assay in HepG2 cells        of TGF-β RI kinase, respectively). It minimally affects a panel        of nine closely related kinases including p38 MAPK (IC₅₀>16 μM).        It is available from Enzo (see website at        enzolifesciences.com/ALX-270-445/alk5-inhibitor-ii/).    -   3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide        (also known as A83-01 from Tocris Bioscience; a selective        inhibitor of TGF-β type I receptor ALK5 kinase, type I        Activin/nodal receptor ALK4 and type I nodal receptor ALK7 (IC50        values can, e.g., be 12, 45 and 7.5 nM respectively);    -   2-(3-(6-Methylpyridine-2-yl)-IH-pyrazol-4-yl)-1,5-naphthyridine        (also known as SJN 2511 from Tocris Bioscience; selective        inhibitor of the TGF-β type I receptor ALK5 (IC50 values can,        e.g., be 0.004 and 0.023 μM for ALK5 autophosphorylation and        ALK5 binding, respectively);    -   4-[4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-IH-imidazol-2-yl]benzamide        (also known as D 4476 from Tocris Bioscience; a selective        inhibitor of casein kinase 1 (CK1) and TGF-β type-1 receptor        (ALK5) that displays greater than 20-fold selectivity over        SAPK2/p38);    -   4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-quinoline (also known as LY        364947 from Tocris Bioscience; a selective inhibitor of TGF-β        type-I receptor (TGF-β R1, TGFR-I, TβR-1, ALK-5) (IC50 values        can, e.g., be 59, 400 and 1400 nM for TGR-β RI, TGF-β RII and        MLK-7K respectively);    -   2-(4-(benzo[d][1,3]dioxol-5-yl)-2-tert-butyl-1H-imidazol-5-yl)-6-methylpyridine        (also known as SB505124, and available from Selleckchem.com; a        selective inhibitor of ALK4 and ALK5 (e.g., with IC50 of 129 nM        and 47 nM, respectively);    -   6-[2-(1,1-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline        (also known as SB 525334 from Sigma-Aldrich; a selective        inhibitor of transforming growth factor-β receptor I (ALK5,        TGF-βRI), with IC50=14.3 nM, for example);    -   2-(5-Chloro-2-fluorophenyl)-4-[(4-pyridyl)amino]pteridine (also        known as SD 208 from Tocris Bioscience; a potent, orally active        ATP-competitive transforming growth factor-β receptor 1        (TGF-βRI) inhibitor, e.g., with IC50=49 nanomolar);    -   4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline        (also known as LDN-193189 from Miltenyi Biotec); and    -   any combination thereof.

The inhibitor that directly or indirectly negatively regulates TGF-betasignaling can, for example, be selected from the group consisting ofSB431542, A83-01, SB-431542, A83-01, SJN-2511, LY-36494, SB-505124,SB-525334, and SD-208. In some embodiments, an inhibitor that directlyor indirectly negatively regulates TGF-beta signaling can inhibit ALK4,ALK5 and/or ALK7. For example, the inhibitor that directly or indirectlynegatively regulates TGF-beta signaling can be A83-01, with thefollowing structure.

The TGF-beta inhibitor can also be in the form of a salt or hydrate ofany of the foregoing compounds.

The time of contacting or mixing a TGFβ receptor inhibitor with apopulation of definitive endodermal cells can vary, for example, fromabout 0.5 days to about 10 days, or from 1 days to about 10 days, orfrom 1.5 days to about 8 days, or from 2 days to about 7 days, or from 2days to about 6 days, or from about 2 days to about 5 days, or about 2days to 4 days.

The time of contacting or mixing a TGFβ receptor inhibitor with apopulation of pancreatic progenitor cells can vary, for example, fromabout 0.5 days to about 10 days, or from 1 days to about 8 days, or from1.5 days to about 6 days, or from 1.5 days to about 5 days, or from 2days to about 4 days, or about 3 days.

TGFβ receptor inhibitors can be used at a variety of concentrations, forexample, at about 0.01 μM to about 10 μM, or from about 0.05 μM to about9 μM, or from about 0.1 μM to about 8 μM, or from about 0.2 μM to about7 μM, or from about 0.3 μM to about 6 μM, or from about 0.4 μM to about5 μM, or from about 0.5 μM to about 3 μM, or about 1 μM.

Various methods for determining if a substance is a TGF-beta inhibitorare available. For example, a cell-based assay can be employed in whichcells are stably transfected with a reporter construct comprising thehuman PAI-1 promoter or Smad binding sites, driving a luciferasereporter gene. Inhibition of luciferase activity relative to controlgroups can be used as a measure of compound activity (De Gouville etal., Br J Pharmacol. 2005 May; 145(2): 166-177). Another example is theALPHASCREEN® phosphosensor assay for measurement of kinase activity(Drew A E et al., Comparison of 2 Cell-Based Phosphoprotein Assays toSupport Screening and Development of an ALK Inhibitor, J Biomol Screen16(2) 164-173, 2011).

Hedgehog Pathway Inhibitors

As shown herein, addition of inhibitors of the hedgehog pathway (withoutaddition of any growth factors) to definitive endoderm cells increasesthe proportion and yield of Pdx1⁺ and/or Nkx6.1⁺ pancreatic progenitorcells. For example, more than about 5%, or more than about 10%, or morethan about 15%, or more than about 20%, or about 25% of the cells werePdx1 positive and/or Nkx6.1 positive.

The hedgehog pathway inhibitors can be added to cells that expressSox17, Foxa2, Cerberus 1 (Cer), C—X—C chemokine receptor type 4 (Cxcr4),or a combination thereof.

To increase the proportion of cells that express markers indicative of apancreatic progenitor cell phenotype, a selected population of cells(e.g., definitive endoderm cells) is contacted or mixed with a hedgehogpathway inhibitor for a time and at a concentration sufficient todifferentiate at least some of the cells into pancreatic progenitorcells.

The time of contacting or mixing a hedgehog pathway inhibitor with theselected population of cells can vary, for example, from about 0.5 daysto about 12 days, or from 1 days to about 10 days, or from 1.5 days toabout 8 days, or from 2 days to about 7 days, or from 2.5 days to about6 days, or from about 3 days to about 5 days, or about 4 days.

Hedgehog pathway inhibitors can be used at a variety of concentrations,for example, at about 0.01 μM to about 10 μM, or from about 0.05 μM toabout 9 μM, or from about 0.1 μM to about 8 μM, or from about 0.3 μM toabout 7 μM, or from about 0.5 μM to about 6 μM, or from about 0.75 μM toabout 5 μM, or from about 1 μM to about 3 μM, or about 2 μM.

A variety of hedgehog pathway inhibitors can be employed. Examples ofhedgehog pathway inhibitors include, for example, LDE 225 (Novartis),cyclopamine, MK-4101 (Merck), GDC-0449 (Genentech), XL-139 (BMS-833923)(Bristol Myers Squibb), PF-04449913 (Pfizer), robotnikinin, andCur-61414 (G-024856).

Notch Inhibitor

As shown herein, addition of inhibitors of Notch to definitive endodermcells increases the proportion and yield of Pdx1⁺ and/or Nkx6.1⁺pancreatic progenitor cells. For example, more than about 5%, or morethan about 10%, or more than about 15%, or more than about 20%, or about25% of the cells were Pdx1 positive and/or Nkx6.1 positive. In additionNotch inhibitors can also increase the proportion and yield of maturepancreatic beta cells.

The Notch inhibitor can operate in any manner that inhibits Notchfunction. For example, the Notch inhibitor can inhibit Notch signaling,inhibit Notch transcription, inhibit Notch translation, or competitivelyinhibit Notch. Examples of Notch inhibitors include gamma secretaseinhibitors, Notch interfering RNA, and dominant negative Notch proteins.

Notch signaling can be modulated by altering the activity of thegamma-secretase complex. This complex cleaves the Notch receptorreleasing the Notch intracellular domain (reviewed in Fortini, NatureReviews Molecular and Cell Biology 3: 673-684 (2002)). Gamma-secretaseinhibitors reduce the level of Notch signaling and lead to effects thatresemble or are identical to the phenotypes produced by loss of functionmutations in Notch genes in a variety of organisms and experimentalsystems (Dovey et al., Journal of Neurochemistry 76:173-181 (2001);Hadland et al., Proceedings of the National Academy of Sciences USA 98:7487-7491 (2001); Doerfler et al., Proceedings of the National Academyof Sciences USA 98: 9312-9317 (2001); Micchelli et al., The FASEBJournal 17: 79-81 (2002)).

A variety of Notch and/or gamma-secretase inhibitors can be employed,including any of the following:

-   -   Compound E (C-E) is a cell permeable, potent, selective,        non-transition state and non-competitive inhibitor of        γ-secretase (IC₅₀=0.3 nM for total β-amyloid) and Notch        processing, which inhibits cell differentiation. At higher        concentrations (20-400 μM), compound E only weakly affects the        presenilase activity. Compound E is commercially available from        a variety of sources, including Enzo Life Sciences        (enzolifesciences.com/ALX-270-415/compound-e/). The structure of        compound E is shown below.

-   -   RO4929097 is a γ secretase inhibitor (available from        Selleckcehem.com) with IC50 of 4 nM, inhibiting cellular        processing of A1340 and Notch with EC50 of 14 nM and 5 nM,        respectively. R04929097 has the following structure:

-   -   DAPT (GSI-IX) is a γ-secretase inhibitor (available from        Sigma-Aldrich) that inhibits Aβ production. DAPT has the        following structure:

-   -   Gamma-Secretase Inhibitor I, which has the following structure:        Z-Leu-Leu-Nle-CHO (Nle=Norleucine) (available from EMD Millipore        (see,        emdmillipore.com/life-science-research/gamma-secretase-inhibitor).    -   Gamma-Secretase Inhibitor II, which is a cell-permeable,        reversible and selective peptidomimetic inhibitor of γ-secretase        (IC₅₀=13 μM for Aβ). It displays only weak inhibitory activity        against calpain II (IC₅₀=100 μM in a purified enzyme assay).        Gamma-Secretase Inhibitor II has the following structure:

In some embodiments, the Notch inhibitor is compound E.

The Notch inhibitor can be used in various concentrations. For example,the Notch inhibitor can be employed at a concentration of about 0.001micromolar to about 200 micromolar, or about 0.01 micromolar to about100 micromolar, or about 0.05 micromolar to about 10 micromolar, orabout 0.1 micromolar in a solution. In a dry formulation, Notchinhibitor can be present in amounts of about 0.01 mg to about 1000 mg,or about 0.1 mg to about 100 mg, or about 1 mg to about 10 mg.

Cells can be incubated in a medium containing a Notch inhibitor (e.g.,compound E (C-E)), for varying amounts of time. For example, the cellscan be incubated in a medium containing a Notch inhibitor until at leastsome of the cells express pancreatic progenitor markers such as Pdx1and/or Nkx6.1⁺. The incubation time can vary, for example, from about0.5 days to about 5 days, or from about 1 day to about 4 days, or fromabout 1 days to about 3 days, or about 2 days.

Alternatively, when converting pancreatic progenitor cells to maturepancreatic beta cells, the cells can be incubated until markers such asinsulin and/or Pdx1. The incubation time for conversion of pancreaticprogenitor cells to mature pancreatic beta cells can vary, for example,from about 2 days to about 20 days, or from about 4 days to about 16days, or from about 6 days to about 15 days, or from about 9 days toabout 14 days, or about 12 days.

BMP4 Signaling Inhibitor

BMP4 is a potent growth factor that has a role in mesodermaldifferentiation, basic body plan formation, and determination of theproximal-distal, left-right, and dorsal-ventral axes. BMP4 elicitsdifferent biological responses depending on the concentration of thesecreted form. For example, high levels of BMP4 in early embryonicdevelopment are associated with commitment to a ventral fate, while lowlevels are associated with a commitment to dorsal neural and musculartissue. Secreted active BMP4 can be inhibited at the extracellular levelby interacting with secreted BMP4 antagonists, such as noggin, chordin,CeM, DAN, and Gremlin.

BMP4 signaling inhibitors are available. For example, the methods andcompositions described herein can include any of the BMP4 inhibitorsdescribed in U.S. Pat. No. 8,507,501, the contents of which are herebyincorporated by reference in their entirety. Another example of a BMP4signaling inhibitor is3-(6-amino-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)phenol (K02288), whichinhibits BMP4 mediated phosphorylation of Smad1/5/8 (Sanvitale et al., ANew Class of Small Molecule Inhibitor of BMP Signaling, PLOS One 8(4):e62721 (Apr. 30, 2013). In some embodiments, the BMP4 signalinginhibitor can be4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline(LDN-193189).

The BMP4 signaling inhibitor can be used in various concentrations. Forexample, the BMP4 signaling inhibitor can be employed at a concentrationof about 0.001 micromolar to about 200 micromolar, or about 0.01micromolar to about 100 micromolar, or about 0.05 micromolar to about 10micromolar, or about 0.1 micromolar in a solution. In a dry formulation,BMP4 signaling inhibitor can be present in amounts of about 0.01 mg toabout 1000 mg, or about 0.1 mg to about 100 mg, or about 1 mg to about10 mg.

Cells can be incubated in a medium containing a BMP4 signaling inhibitor(e.g., LDN-193189), for varying amounts of time. For example, the cellscan be incubated in a medium containing a BMP4 signaling inhibitor untilat least some of the cells express pancreatic progenitor markers such asPdx1⁺ and/or Nkx6.1⁺. The incubation time can vary, for example, fromabout 0.5 days to about 5 days, or from about 1 day to about 4 days, orfrom about 1 day to about 3 days, or about 2 or 3 days.

Activators of Protein Kinase C

Protein kinase C is an enzyme that regulates the function of otherproteins by phosphorylation of serine and/or threonine hydroxy groups onthose proteins.

Activators of protein kinase C include phorbol 12,13-dibutyrate (PBDu),Bryostatin, FR 236924, (−)-Indolactam V, PEP 005, phorbol12,13-dibutyrate, phorbol 12-myristate13-acetate, PKC fragment(530-558), pseudo RACK1, SC-10, SC-9, and combinations thereof. Theseprotein kinase C activators are available from Tocris (see website attocris.com/pharmacologicalBrowser.php?ItemId=164170&Type=Activators#.U8mK601 OU50.

The protein kinase C activator can be used in various concentrations.For example, the protein kinase C activator can be employed at aconcentration of about 0.01 nanomolar to about 3 nanomolar, or about0.05 nanomolar to about 1 nanomolar, or about 0.1 nanomolar to about 0.5nanomolar, or about 0.2 nanomolar in a solution. In a dry formulation,protein kinase C activator can be present in amounts of about 0.01 mg toabout 1000 mg, or about 0.1 mg to about 100 mg, or about 1 mg to about10 mg.

Cells can be incubated in a medium containing a protein kinase Cactivator (e.g., phorbol 12,13-dibutyrate), for varying amounts of time.For example, the cells can be incubated in a medium containing a proteinkinase C activator until at least some of the cells express pancreaticprogenitor markers such as Pdx1⁺ and/or Nkx6.1⁺. The incubation time canvary, for example, from about 0.5 days to about 7 days, or from about 1day to about 5 days, or about 2 to 4 days.

Expansion of Pancreatic Progenitor Cells

Pancreatic progenitor cells can be expanded using the same procedures asfor expansion of endodermal cells. However the pancreatic progenitorcell colonies easily detached from culture plates when such procedureswere employed. Removal of the WNT activator, and increased amounts ofgrowth factors (e.g., EGF) obviated this problem and improved pancreaticprogenitor cell expansion. Under these optimized culture conditions,pancreatic progenitor cells were expanded more than two hundred millionfold with an approximate doubling time of 3 days for up to 14 passages.Moreover, the cells maintained their bi-potent progenitor identity asevidenced by the presence of PDX1 and NKX6.1 double-positive cells atpassage 12.

Pancreatic progenitor cells are ideal for transplantation and treatmentdue to their potential for sustained proliferation and properdifferentiation. The expansion methods and compositions described hereinfacilitate production of sufficient numbers of cells to betherapeutically useful.

Pancreatic Beta Cells

Pancreatic beta cells can be identified by their expression of insulin,C-peptide, PDX1, NKX6.1, NEUROD1, NKX2.2, glucagon, or any combinationthereof. The procedures and compositions described herein can convertmore than about 1%, or more than about 2%, or more than about 3%, ormore than about 4%, or about 5% of pancreatic progenitor cells topancreatic beta cells as illustrated by expression of insulin,C-peptide, PDX1, NKX6.1, NEUROD1, NKX2.2, glucagon, or any combinationthereof.

Pancreatic beta cells can be generated from pancreatic progenitor cellsby incubation in media that includes a TGFβ receptor inhibitor (e.g.A83-01), a polyADP-ribose synthetase inhibitor (e.g. nicotinamide), anagonist of glucocorticoid receptor (e.g., dexamethasone), a NotchSignaling Inhibitor (e.g., Compound E), and combinations thereof.Conversion of pancreatic progenitor cells to pancreatic beta cells isimproved by inclusion of all four of the foregoing agents in the culturemedium. Conversion of pancreatic progenitor cells to pancreatic betacells is also improved by addition of vitamin C, an activator ofadenylyl cyclase (e.g. Forskolin), an agonist of Glucagon-like Peptide-1(e.g., Exendin-4), a Ca²⁺ channel agonist (e.g. BayK-8644), or anycombination thereof to the culture medium.

Pancreatic beta cells can also be generated in cell culture mediacontaining laminin, nicotinamide, and B27 etc. as previously reported(Schroeder et al., 2006). As described herein, small molecules such as2-phospho-L-ascorbic acid and inhibitors of a p38 mitogen-activatedprotein (MAP) kinase strongly and synergistically increased the numberof insulin⁺/Pdx1⁺ cells in the mixture. For example, the SB203580inhibitor of a p38 mitogen-activated protein (MAP) kinase and/or2-phospho-L-ascorbic acid promote pancreatic beta-cell maturation andincreased the number of insulin⁺/Pdx1⁺ cells in the mixture.

Pancreatic beta-like cells can be generated by successive exposure ofless differentiated cells such as pancreatic progenitor cells todifferent media using a process referred to herein as Protocol 2. In afirst step, pancreatic endodermal progenitor cells were cultured in thepancreatic maturation media containing heparin, zinc salts, a TGF-βinhibitor (e.g., Alk5 inhibitor), a BMP4 signaling inhibitor (e.g.,LDN-193189), a T3 thyroid hormone, a Notch Signaling Inhibitor (e.g.,Compound E), a Ca²⁺ channel agonist (e.g. BayK-8644), vitamin C, andcombinations thereof to generate a first population of partiallydifferentiated pancreatic beta-like cells. In a second step, this firstpopulation of cells is contacted with a composition containing heparinzinc salts, a TGF-β inhibitor (e.g., Alk5 inhibitor), a BMP4 signalinginhibitor (e.g., LDN-193189), a T3 thyroid hormone, cysteine, ananti-oxidant (e.g., vitamin E or a derivative thereof such as Trolox),an Axl kinase inhibitor (e.g., R428), a Ca²⁺ channel agonist (e.g.BayK-8644), vitamin C, and combinations thereof to generate a secondpopulation of pancreatic beta-like cells. In a third step, the secondpopulation of cells is cultured to generate 3D aggregates underlow-attachment plates. FIG. 12A illustrates one way to perform protocol2 to generate pancreatic beta-like cells (cPB) from pancreaticendodermal progenitor cells.

Several of the factors employed to convert pancreatic progenitor cellsto pancreatic beta cells have been described in the foregoing sections.Further information about other factors useful for converting pancreaticprogenitor cells to pancreatic beta cells is described below.

PolyADP-Ribose Synthetase Inhibitors

PolyADP-ribose synthetase is also referred to as polyADP-ribosepolymerase (PARP). PolyADP-ribose synthetase is an enzyme that catalyzesthe poly-ADP ribosylation reaction; this enzyme has an important role inDNA repair or transcriptional regulation.

As shown herein, addition of polyADP-ribose synthetase inhibitor topancreatic progenitor cells increases the proportion and yield of cellsthat express genes indicative of the pancreatic beta cell phenotype. Toincrease the proportion of cells that convert to pancreatic beta cells(and express markers indicative of a pancreatic beta cell phenotype), aselected population of cells (e.g., pancreatic progenitor cells) iscontacted or mixed with one or more polyADP-ribose synthetase inhibitorsfor a time and at a concentration sufficient to differentiate at leastsome of the cells into pancreatic beta cells.

Examples of a polyADP-ribose synthetase inhibitor include nicotinamide,3-aminobenzamide, 1,5-isoquinolinediol and combinations thereof.

The polyADP-ribose synthetase inhibitor can be used in variousconcentrations. For example, the polyADP-ribose synthetase inhibitor canbe employed at a concentration of about 0.1 millimolar to about 100millimolar, or about 1 millimolar to about 50 millimolar, or about 3millimolar to about 30 millimolar, or about 5 micromolar to about 15millimolar, or about 10 millimolar in a solution. In a dry formulation,polyADP-ribose synthetase inhibitor can be present in amounts of about 1mg to about 1000 mg, or about 10 mg to about 100 mg.

Cells can be incubated in a medium containing a polyADP-ribosesynthetase inhibitor (e.g., nicotinamide), for varying amounts of time.For example, the cells can be incubated in a medium containing apolyADP-ribose synthetase inhibitor until at least some of the cellsexpress pancreatic beta cell markers such as Pdx1⁺, insulin, C-peptide,or a combination thereof. The incubation time can vary, for example,from about 0.5 days to about 5 days, or from about 1 day to about 4days, or from about 1 days to about 3 days, or about 2 or 3 days.

p38 Mitogen-Activated Protein (MAP) Kinase Inhibitors

Inhibitors of a p38 mitogen-activated protein (MAP) kinase can be addedto Pdx⁺/Nkx6.1⁺ pancreatic progenitor cells to generate pancreatic betacells.

To increase the proportion of cells that convert to pancreatic betacells (and express markers indicative of a pancreatic beta cellphenotype), a selected population of cells (e.g., pancreatic progenitorcells) is contacted or mixed with p38 mitogen-activated proteininhibitors for a time and at a concentration sufficient to differentiateat least some of the cells into pancreatic beta cells.

Examples of p38 mitogen-activated protein inhibitors that can be used inthe methods and compositions described herein include, but are notlimited to, p38 inhibitors SB203580 (i.e.,4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole),SB202190 (i.e., 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole), SB239063 (i.e.,trans-1-(-Hydroxycyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyrimidin-4-yl)imidazole),SB220025(5-(2-Aminopyrimidin-4-yl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazoletrihydrochloride), PD169316(4-(4-Fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole),RPR200765A([2-[4-(4-fluorophenyl)-5-pyridin-4-yl-1H-imidazol-2-yl]-5-methyl-1,3-dioxan-5-yl]-morpholin-4-ylmethanone;methanesulfonic acid) AMG 548(2-[[(2S)-2-amino-3-phenylpropyl]amino]-3-methyl-5-naphthalen-2-yl-6-pyridin-4-ylpyrimidin-4-one),BIRB-796(1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[4-(2-morpholin-4-ylethoxy)naphthalen-1-yl]urea),SCID-469(6-Chloro-5-[[(2R,5S)-4-[(4-fluorophenyl)methyl]-2,5-dimethyl-1-piperazinyl]carbonyl]-N,N,1-trimethyl-α-oxo-1H-Indole-3-acetamide),SCID-323 and VX-702 disclosed in Nikas et al. (Curr Opin Drug DiscovDevel. 8, 421-430. (2005)), FR167653 disclosed in Yamamoto et al. (Eur.J. Pharmacol 314, 137-142 (1996)), and combinations thereof.

The time of contacting or incubating p38 mitogen-activated proteininhibitors with pancreatic progenitor cells can vary, for example, fromabout 1 days to about 20 days, or from 2 days to about 15 days, or from3 days to about 13 days, or from 4 days to about 12 days, or from 5 daysto about 11 days, or from about 6 days to about 11 days, or for about 9days.

For example, inhibitors of a p38 mitogen-activated protein (MAP) kinasecan be added to a selected cell population (pancreatic progenitor cells)while directing the cells to differentiate into pancreatic beta cells.Inhibitors of a p38 mitogen-activated protein (MAP) kinase such asSB203580 can be employed at a variety of concentrations, for example, atabout 0.1 μM to about 500 μM, or from about 0.5 μM to about 400 μM, orfrom about 1 μM to about 200 μM, or from about 2 μM to about 100 μM, orfrom about 2.5 μM to about 50 μM, or from about 3 μM to about 25 μM, orfrom about 4 μM to about 10 μM, or about 5 μM.

Agonists of Glucagon-like Peptide-1

Glucagon-like peptide-1 agonists are a group of medications that mimicthe actions of glucagon-like peptide-1 (GLP-1). GLP-1 is one of severalnaturally occurring incretin compounds that affect the body after theyare released from the gut during digestion. Because of its name, GLP-1might seem to act like glucagon that increases glucose production by theliver and raises glucose levels. Instead, GLP-1 lowers both glucose andglucagon levels. Despite their different actions, GLP-1 and glucagon areboth derived from the same parent compound called proglucagon, hence thesimilarity in names.

To increase the proportion of cells that convert to pancreatic betacells (and express markers indicative of a pancreatic beta cellphenotype), a selected population of cells (e.g., pancreatic progenitorcells) is contacted or mixed with GLP-1 agonists for a time and at aconcentration sufficient to differentiate at least some of the cellsinto pancreatic beta cells.

Examples of GLP-1 agonists that can be used in the methods andcompositions described herein include, but are not limited to,Extendin-4, exenatide (Byetta/Bydureon), liraglutide (Victoza),lixisenatide (Lyxumia), albiglutide (Eperzan), dulaglutide, andcombinations thereof.

The time of contacting or incubating GLP-1 agonists with pancreaticprogenitor cells can vary, for example, from about 5 days to about 40days, or from 8 days to about 35 days, or from 10 days to about 30 days,or from 12 days to about 25 days, or for about 8 to 12 days followed bya second incubation of about 8 days to about 12 days.

For example, GLP-1 agonists can be added to a selected cell population(pancreatic progenitor cells) while directing the cells to differentiateinto pancreatic beta cells. GLP-1 agonists such as Extendin-4 can beemployed at a variety of concentrations, for example, at about 5 ng/mlto about 200 ng/ml, or from about 10 ng/ml to about 175 ng/ml, or fromabout 15 ng/ml to about 150 ng/ml, or from about 20 ng/ml to about 150ng/ml, or from about 25 ng/ml to about 125 ng/ml, or from about 30 ng/mlto about 100 ng/ml, or from about 35 ng/ml to about 80 ng/ml, or fromabout 40 ng/ml to about 60 ng/ml, or about 50 ng/ml.

Adenylyl Cyclase Activators

Adenylyl cyclase is an enzyme that employs pyrophosphate to catalyze theconversion of ATP to 3′,5′-cyclic ATP, which is a second messengerinvolved in several biological processes. Although there are sixdistinct classes of adenylyl cyclase enzymes, all catalyze the samereaction.

To increase the proportion of cells that convert to pancreatic betacells (and express markers indicative of a pancreatic beta cellphenotype), a selected population of cells (e.g., pancreatic progenitorcells) is contacted or mixed with one or more adenylyl cyclaseactivators for a time and at a concentration sufficient to differentiateat least some of the cells into pancreatic beta cells.

Examples of adenylyl cyclase activators that can be used in the methodsand compositions described herein include, but are not limited to,Forskolin, NKH 477, PACAP 1-27, PACAP 1-38, and combinations thereof.Each of these adenylyl cyclase activators is available from Tocris (see,e.g., the website at tocris.com/pharmacologicalBrowser. php?ItemId=5136&Type=Activators#.U8kzv01OU5s).

The time of contacting or incubating adenylyl cyclase activators withpancreatic progenitor cells can vary, for example, from about 5 days toabout 40 days, or from 8 days to about 35 days, or from 10 days to about30 days, or from 12 days to about 25 days, or for about 8 to 12 daysfollowed by a second incubation of about 8 days to about 12 days.

For example, adenylyl cyclase activators can be added to a selected cellpopulation (pancreatic progenitor cells) while directing the cells todifferentiate into pancreatic beta cells. Adenylyl cyclase activatorssuch as Forskolin can be employed at a variety of concentrations, forexample, at about 0.1 μM to about 500 μM, or from about 0.5 μM to about400 μM, or from about 1 μM to about 200 μM, or from about 2 μM to about100 μM, or from about 2.5 μM to about 50 μM, or from about 3 μM to about25 μM, or from about 5 μM to about 15 μM, or about 10 μM.

Glucocorticoid Receptor Agonist

Glucocorticoid receptors bind cortisol and glucocorticoids and regulatetranscription of genes involved in development, metabolism, and immuneresponses. Glucocorticoid receptors are expressed in almost every cellin the body.

To increase the proportion of cells that convert to pancreatic betacells (and express markers indicative of a pancreatic beta cellphenotype), a selected population of cells (e.g., pancreatic progenitorcells) is contacted or mixed with glucocorticoid receptor agonists for atime and at a concentration sufficient to differentiate at least some ofthe cells into pancreatic beta cells.

Examples of glucocorticoid receptor agonists that can be used in themethods and compositions described herein include, but are not limitedto, dexamethasone, corticosterone, fluticasone propionate, GSK 9027,methylprednisolone, mometasone furoate, and combinations thereof.

The time of contacting or incubating glucocorticoid receptor agonistswith pancreatic progenitor cells can vary, for example, from about 5days to about 40 days, or from 8 days to about 35 days, or from 10 daysto about 30 days, or from 12 days to about 25 days, or for about 8 to 12days followed by a second incubation of about 8 days to about 12 days.

For example, glucocorticoid receptor agonists can be added to a selectedcell population (pancreatic progenitor cells) while directing the cellsto differentiate into pancreatic beta cells. Glucocorticoid receptoragonists such as dexamethasone can be employed at a variety ofconcentrations, for example, at about 0.1 μM to about 500 μM, or fromabout 0.5 μM to about 400 μM, or from about 1 μM to about 200 μM, orfrom about 2 μM to about 100 μM, or from about 2.5 μM to about 50 μM, orfrom about 3 μM to about 25 μM, or from about 5 μM to about 15 μM, orabout 10 μM.

Antioxidants

An antioxidant is a molecule that inhibits the oxidation of othermolecules. Oxidation is a chemical reaction involving the loss ofelectrons or an increase in oxidation state. Oxidation reactions canproduce free radicals. In turn, these radicals can start chainreactions. Examples of antioxidants include as glutathione, vitamin C,vitamin A, vitamin E, trolox, catalase, superoxide dismutase and variousperoxidases.

One example of an antioxidant that can be used in the compositions andmethods described herein is Trolox. Trolox(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) is awater-soluble analog of vitamin E available from Hoffman-LaRoche. It isan antioxidant like vitamin E and it can be used in biological orbiochemical applications to reduce oxidative stress or damage.

The antioxidants can be can be added to a cell population (e.g.,pancreatic progenitor cells) while directing the cells to differentiateinto pancreatic beta cells. Antioxidants such as trolox can be employedat a variety of concentrations, for example, at about 0.1 μM to about500 μM, or from about 0.5 μM to about 400 μM, or from about 1 μM toabout 200 μM, or from about 2 μM to about 100 μM, or from about 2.5 μMto about 50 μM, or from about 3 μM to about 25 μM, or from about 5 μM toabout 15 μM, or about 10 μM.

Axl Kinase Inhibitors

Examples of Axl kinase inhibitors that can be used in the compositionsand methods described herein include R428, 549076, LY2801653, MP-470(Amuvatinib), SKI-606 (Bosutinib), MGCD 265, MGCD516, ASP2215, XL184(Cabozantinib), BMS-777607 (ASLAN 002), GSK1363089/XL880 (Foretinib),SGI-7079, DP3975, NPS-1034, LDC1267, NA80x1, YW327.6S2 antibodies,GL21.T aptamers, and combinations thereof.

R428 (BGB324) is an inhibitor of Axl with IC₅₀ of 14 nM, demonstratingthat R428 is more than 100-fold selective for Axl than for Abl.Selectivity of R428 for Axl is also greater than Mer and Tyro3(50-to-100-fold more selective) and InsR, EGFR, HER2, and PDGFRβ(100-fold more selective). The structure of R428 is shown below:

S49076(3-[(3-{[4-(4-Morpholinylmethyl)-1H-pyrrol-2-yl]methylene}-2-oxo-2,3-dihydro-1H-indol-5-yl)methyl]-1,3-thiazolidine-2,4-dione)is a novel, potent inhibitor of MET, AXL/MER, and FGFR1/2/3. S49076potently blocks cellular phosphorylation of MET, AXL, and FGFRs andinhibits downstream signaling in vitro and in vivo. In cell models,S49076 inhibits the proliferation of MET- and FGFR2-dependent gastriccancer cells, blocks MET-driven migration of lung carcinoma cells, andinhibits colony formation of hepatocarcinoma cells expressing FGFR1/2and AXL. See, Burbridge et al., Mol Cancer Ther 12(9):1749-62 (September2013).

LY2801653 is a type-II ATP competitive, slow-off inhibitor of METtyrosine kinase. The K_(i) value of LY2801653 for MET is 2 nM. However,preclinical testing also has shown that LY2801653 inhibits several otherreceptor tyrosine oncokinases including MST1R, FLT3, AXL, MERTK, TEK,ROS1, and DDR1/2 and the serine/threonine kinases MKNK1/2. The structureof LY2801653 is shown below.

LY2801653 can be obtained from a variety of sources such as MedChemExpress (see website atmedchemexpress.com/LY2801653.html?gclid=COnZ-MLq28YCFY4AaQodC7ALeQ),APExBio (see website atapexbt.com/ly2801653.html?gclid=CImtkffq28YCFQipaQodFWEBmQ), or KareBayBiochem (see website at karebaybio.com/products/ly2801653.html?gclid=CI7IyKrr28YCFQqsaQodUJIHog).

Amuvatinib (MP-470) is a potent and multi-targeted inhibitor of c-Kit,PDGFRα and Flt3 with IC₅₀ of 10 nM, 40 nM and 81 nM, respectively.MP-470 (1 μM) also inhibits tyrosine phosphorylation of AXL. Thestructure of MP-470 is shown below.

Bosutinib is selective for Src over non-Src family kinases with an IC50of 1.2 nM, and potently inhibits Src-dependent cell proliferation withan IC50 of 100 nM. Bosutinib is also being tested as an Axl kinaseinhibitor (Wu et al., Oncotarget 5(20): 9546-9563 (October 2014)). Thestructure of Bosutinib is shown below.

MGCD-265 is a multi-target inhibitor of receptor tyrosine kinases. Inc-Met-driven or non-c-Met-driven mice xenograft models of MKN45, U87MG,MDA-MB-231, COLO205, and A549 tumor cells, MGCD-265 (20 mg/kg-60 mg/kg)inhibits tumor growth and c-Met signaling. The structure of MGCD-265 isshown below.

MGCD-265 is available from Selleck.com (see website atselleckchem.com/products/MGCD-265.html).

MGCD516 is a small molecule that has demonstrated potent inhibition of aclosely related spectrum of tyrosine kinases including Trk, RET and DDR,which are key regulators of signaling pathways that lead to cell growth,survival and tumor progression. It is made by Mirati Therapeutics.

Gilteritinib, also known as (ASP2215), is a potent FLT3/AXL inhibitor,which showed potent anti-leukemic activity against AML with either orboth FLT3-ITD and FLT3-D835 mutations. Among the 78 tyrosine kinasestested in vitro, ASP2215 inhibited FLT3, LTK, ALK, and AXL kinases byover 50% at 1 nM. ASP2215 is available from ActiveBiochem.com. Thestructure of ASP2215 is shown below.

The Axl kinase inhibitors can be added to a cell population (e.g.,pancreatic progenitor cells) while directing the cells to differentiateinto pancreatic beta cells. Axl kinase inhibitors such as R428 can beemployed at a variety of concentrations, for example, at about 0.01 μMto about 50 μM, or from about 0.05 μM to about 40 μM, or from about 0.1μM to about 20 μM, or from about 0.5 μM to about 10 μM, or from about 1μM to about 5 μM, or about 2 μM.

T3 Thyroid Hormone

The thyroid hormones, triiodothyronine (T3) and its prohormone,thyroxine (T4), are tyrosine-based hormones produced by the thyroidgland that are primarily responsible for regulation of metabolism. Thesehormones bind iodine. The structure of the T3 thyroid hormone is asfollows:

Thyroid hormones such as the T3 thyroid hormone can be employed at avariety of concentrations, for example, at about 0.01 μM to about 100μM, or from about 0.05 μM to about 50 μM, or from about 0.1 μM to about10 μM, or from about 0.2 μM to about 7 μM, or from about 0.5 μM to about5 μM, or from about 0.7 μM to about 2 μM, or about 1 μM.

Ca²⁺ Channel Agonists

Ca²⁺ channel agonists can increase calcium influx into calcium channelsof excitable tissues. To increase the proportion of cells that convertto pancreatic beta cells (and express markers indicative of a pancreaticbeta cell phenotype), a selected population of cells (e.g., pancreaticprogenitor cells) is contacted or mixed with Ca²⁺ channel agonists for atime and at a concentration sufficient to differentiate at least some ofthe cells into pancreatic beta cells.

Examples of Ca²⁺ channel agonists that can be used in the methods andcompositions described herein include, but are not limited to, BayK-8644(3-pyridinecarboxylic acid,1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl), methylester), CGP 28392(2-methyl-3-methoxycarbonyl-4-(2′-difluoromethoxyphenyl)-5-oxo-1,4,5,7-tetrahydrofuro(3,4-b)pyridine),calcitriol, 1-ethyl-2-benzimidazolinone, FPL 64176 (methyl2,5-dimethyl-4-(2-(phenylmethyl)benzoyl)-1H-pyrrole-3-carboxylate), PN202-791(4-isopropyl-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro-3-pyridinecarboxylate),SAN 202791, and combinations thereof.

The time of contacting or incubating Ca²⁺ channel agonists withpancreatic progenitor cells can vary, for example, from about 5 days toabout 40 days, or from 8 days to about 35 days, or from 10 days to about30 days, or from 12 days to about 25 days, or for about 8 to 12 daysfollowed by a second incubation of about 8 days to about 12 days.

For example, Ca²⁺ channel agonists can be added to a selected cellpopulation (pancreatic progenitor cells) while directing the cells todifferentiate into pancreatic beta cells. Ca²⁺ channel agonists such asBayK-8644 can be employed at a variety of concentrations, for example,at about 0.05 μM to about 200 μM, or from about 0.1 μM to about 100 μM,or from about 0.2 μM to about 50 μM, or from about 0.3 μM to about 25μM, or from about 0.5 μM to about 10 μM, or from about 1 μM to about 5μM, or from about 1.5 μM to about 4 μM, or about 2 μM.

Basement Membrane Proteins

Basement membrane proteins can increase calcium influx into calciumchannels of excitable tissues. To increase the proportion of cells thatconvert to pancreatic beta cells (and express markers indicative of apancreatic beta cell phenotype), a selected population of cells (e.g.,pancreatic progenitor cells) is contacted or mixed with one or morebasement membrane proteins for a time and at a concentration sufficientto differentiate at least some of the cells into pancreatic beta cells.

Examples of basement membrane proteins that can be used in the methodsand compositions described herein include, but are not limited to,laminin, collagens, fibrillins, integrins, entactins, dystroglcans, andcombinations thereof.

The time of contacting or incubating one or more basement membraneproteins with pancreatic progenitor cells can vary, for example, fromabout 1 days to about 30 days, or from about 2 days to about 25 days, orfrom about 3 days to about 20 days, or from about 5 days to about 15days, or for about 7 to 12 days.

For example, basement membrane proteins can be added to a selected cellpopulation (pancreatic progenitor cells) while directing the cells todifferentiate into pancreatic beta cells. Glucocorticoid receptoragonists such as dexamethasone can be employed at a variety ofconcentrations, for example, at about 0.05 μM to about 200 μM, or fromabout 0.1 μM to about 100 μM, or from about 0.2 μM to about 50 μM, orfrom about 0.3 μM to about 25 μM, or from about 0.5 μM to about 10 μM,or from about 1 μM to about 5 μM, or from about 1.5 μM to about 4 μM, orabout 2 μM.

Other Agents

The compositions and methods that can be used for converting pancreaticendodermal progenitor cells to pancreatic beta-like cells can alsoinclude heparin zinc salts, and/or cysteine.

The heparin can be can be added to a selected cell population(pancreatic progenitor cells) while directing the cells to differentiateinto pancreatic beta cells. Heparin can be employed at a variety ofconcentrations, for example, at about 0.05 μg/ml to about 200 μg/ml, orfrom about 0.1 μg/ml to about 100 μg/ml, or from about 0.5 μg/ml toabout 70 μg/ml, or from about 1 μg/ml to about 50 μg/ml, or from about 2μg/ml to about 20 μg/ml, or from about 5 μg/ml to about 15 μg/ml, orabout 10 μg/ml.

The zinc salts that can be employed in the compositions and methodsdescribed herein include any pharmaceutically acceptable zinc salt.Examples, include zinc acetate, zinc benzoate, zinc borate, zincbromide, zinc carbonate, zinc citrate, zinc chloride, zincglycerophosphate, zinc hexafluorosilicate, zinc dl-lactate (trihydrate),zinc nitrate, zinc phenolsulfonate, zinc silicate, zinc alkanoateshaving 8-18 carbon atoms, zinc salicylate, zinc stannate, zinc sulfate,zinc tannate, zinc tartrate, zinc titanate, zinc tetrafluoroborate, zincoxide, zinc peroxide, zinc hydroxide or combinations thereof. The zinccompounds may be used singly or in admixture. Glycine can be present tostabilize the zinc salts. For example, the zinc salts can be zincsulfate or zinc chloride.

The zinc salts can be added to a cell population (e.g., pancreaticprogenitor cells) while directing the cells to differentiate intopancreatic beta cells. Zinc salts such as zinc sulfate can be employedat a variety of concentrations, for example, at about 0.1 μM to about500 μM, or from about 0.5 μM to about 400 μM, or from about 1 μM toabout 200 μM, or from about 2 μM to about 100 μM, or from about 2.5 μMto about 50 μM, or from about 3 μM to about 25 μM, or from about 5 μM toabout 15 μM, or about 10 μM.

Cysteine can be added to a cell population (e.g., pancreatic progenitorcells) while directing the cells to differentiate into pancreatic betacells at concentrations such as about 0.01 mM to about 10 mM, or fromabout 0.05 mM to about 9 mM, or from about 0.1 mM to about 8 mM, or fromabout 0.2 mM to about 7 mM, or from about 0.3 mM to about 6 mM, or fromabout 0.4 mM to about 5 mM, or from about 0.5 mM to about 3 mM, or about1 mM.

The disclosure provides a method for directly differentiating pancreaticbeta cell equivalents or pancreatic beta-like cells directly from stemcells (e.g., embryonic stem cells) in a reliable and reproducible mannerthat quickly and efficiently yield substantial numbers of pancreaticbeta cell equivalents or pancreatic beta-like cells. The pancreatic betacell equivalents or pancreatic beta-like cells have physical andfunctional characteristics of pancreatic beta cells and are, therefore,referred to as pancreatic beta cells herein. It should be noted that theterms “pancreatic beta cells” and “pancreatic beta-like cells” are usedinterchangeably herein when referring to the cells produced as a resultof the disclosed methods. These cells provide a number of potentialbenefits, including a screening tool useful in drug screens, toxicologystudies, and the like, a basic research tool to illuminate criticalparameters of normal and diseased pancreatic beta cell function, asproducts useful in methods of identifying novel therapeutic targets, astools to test bioengineering devices (including cell encapsulationdevices), and as tools to develop organs on chips. The organ-buildingapplication is expected to involve, for example, the building ofpancreatic islets by combining the pancreatic beta cells with other celltypes, e.g., endothelial cells, mesenchymal cells, neural cells, immunecells, other hormone-producing cells, non-pancreatic tissues and organs,e.g., liver, fat, muscle, and the like, and as a cell therapy productfor subjects, such as patients, with diabetes.

The disclosed methods provide for the production of functionalpancreatic insulin-producing pancreatic beta cells that function likeendogenous human pancreatic beta cells residing in human islets fromhuman pluripotent stem cells by addition of vitamin C and the BayK-8644compound (i.e., methyl2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-1,4-dihydropyridine-3-carboxylate) at late stages of differentiation (FIG. 15). The disclosed methodsand cells provide advantages over the state of the art in the form of arelatively pure generation of pancreatic progenitor cells by exposingstem cells (e.g., embryonic stem cells) to a refined combination andconcentration of known factors. In addition, disclosed are two compounds(i.e., vitamin C and BayK-8644) that allow for the improved productionof functional pancreatic beta cells in a fast and efficient manner. Inaddition, the disclosed methods yield pancreatic beta cells that exhibitenhanced survival and function as transplanted pancreatic beta cells,and that exhibit superior pancreatic beta cell phenotype for in vitrouse. These advantages enable the use of the disclosed method to producepancreatic beta cells exhibiting immediate functionality aftertransplantation, in contrast to methodologies known in the art fortransplanting progenitor cells, followed by in vivo maturation of thosecells over several months.

The disclosed methods are fast and efficient and result in up to 75%pancreatic beta cells within three weeks of direct differentiation ofhuman pluripotent stem cells. The first stages of direct differentiationutilize a procedure for exactly recapitulating human development byomitting BMP inhibitors during pancreas induction. The time of EGF/KGFexposure has been further extended to 24-48 hours to increase thepercentage PDX1/NKX6.1-positive progenitor cells. Of note, progenitorscan be expanded in this media without the unwanted induction ofprecocious endocrine differentiation marked by early NGN3 expression.The disclosed method can contain optimized combinations andconcentrations of factors known to induce endocrine differentiation. Animproved cocktail can include or consist of DMEM base media containingnon-essential amino acids, glutamine, heparin, cysteine, zinc, ALK(anaplastic lymphoma kinase) and BMP (bone morphogenetic protein)inhibition, T3 thyroid hormone (i.e., triiodothyronine) and the gammasecretase inhibitor XX. The protocols disclosed herein for producingfunctional pancreatic beta cells are faster and more efficient thanprotocols known in the art; moreover, the addition of vitamin C andBayK-8644 results in the generation of pancreatic beta cells exhibitingimproved functionality. These ‘true’ pancreatic beta cells exhibit keyfeatures of bona fide pancreatic beta cells, e.g., several-fold increasein insulin secretion upon glucose stimulation, ultrastructuralmorphology, and functionality after short-term transplantation (FIG.16).

In some embodiments, a functional pancreatic beta cell produced by thesubject methods exhibits a 1-7-fold increase in insulin secretion uponstimulation with glucose. In some embodiments, a functional pancreaticbeta cell produced by the subject methods exhibits a 1-10-fold increasein insulin secretion upon stimulation with glucose. In some embodiments,a functional pancreatic beta cell produced by the subject methodsexhibits a 1.5-10 fold increase in insulin secretion upon stimulationwith glucose (e.g., a 1.5-7-fold, a 2-10-fold, a 2-7-fold, a 3-10-fold,or a 3-7-fold increase in insulin secretion upon stimulation withglucose). In some embodiments, a functional pancreatic beta cellproduced by the subject methods exhibits a 1.5-fold or more increase ininsulin secretion upon stimulation with glucose (e.g., a 2-fold or more,a 3-fold or more, a 5-fold or more, or a 10-fold or more increase ininsulin secretion upon stimulation with glucose).

Consistent with the foregoing descriptions, an aspect of the disclosureis drawn to a method of producing a pancreatic beta cell from a stemcell comprising: (a) exposing a stem cell to Epidermal Growth Factorand/or Keratinocyte Growth Factor for 12-72 hours (e.g., 12-60, 12-48,12-36, 24-72, or 24-60 hours, e.g., in some cases 24-48 hours) underconditions suitable for cell culture growth, thereby maintaining aprogenitor cell; (b) incubating the progenitor cell in a culture mediumcomprising heparin, cysteine, zinc, ALK inhibitor, BMP inhibitorLDN-193189, T3 thryroid hormone and secretase inhibitor XX to yield acell in culture; and (c) adding to the cell in culture viamin C andBayK-8644, thereby producing a functional pancreatic beta cell.

In some embodiments, Epidermal Growth Factor of step (a) is at aconcentration in a range of from 10-300 ng/ml (e.g., 10-200, 10-150,10-100, 10-75, 20-300, 20-200, 20-150, 20-100, 20-75, 25-300, 25-200,25-150, 25-100, or 25-75 ng/ml, e.g., 50 ng/ml).

In some embodiments, Keratinocyte Growth Factor of step (a) is at aconcentration in a range of from 10-300 ng/ml (e.g., 10-200, 10-150,10-100, 10-75, 20-300, 20-200, 20-150, 20-100, 20-75, 25-300, 25-200,25-150, 25-100, or 25-75 ng/ml, e.g., 50 ng/ml).

In some cases, the exposing of step (a) is in a culture medium (e.g.,DMEM), comprising 0.1-5 mM glutamine (e.g., 0.1-4, 0.1-3, 0.5-5, 0.5-4,0.5-3, 1-5, 1-4, 1-3, or 1.5-2.5 mM glutamine, e.g., 2 mM glutamine) or0.05-2.5X GlutaMAX™ (e.g., 0.05-2X, 0.05-1.5X, 0.2-2.5X, 0.2-2X,0.2-1.5X, 0.5-2.5X, 0.5-2X, 0.5-1.5X, or 0.75-1.25X GlutaMAX, e.g., 1XGlutaMAX), 0.1-5X non-essential amino acids (e.g., 0.1-4X, 0.1-3X,0.5-5X, 0.5-4X, 0.5-3X, 0.5-2X, 0.8-5X, 0.8-4X, 0.8-3X, 0.8-2X,0.8X-1.5X, or 0.8X-1.2X non-essential amino acids), and 0.1-5X B27supplement (e.g., 0.1-4X, 0.1-3X, 0.5-5X, 0.5-4X, 0.5-3X, 0.5-2X,0.8-5X, 0.8-4X, 0.8-3X, 0.8-2X, 0.8X-1.5X, or 0.8X-1.2X B27 supplement).In some cases, the exposing of step (a) is in a culture medium (e.g.,DMEM), comprising 2 mM glutamine or 1X GlutaMAX™.

In some embodiments, heparin of step (b) is at a concentration in arange of from 2-20 μg/ml (e.g., 2-15, 5-20, or 5-10 μg/ml, e.g., 10μg/ml). In some embodiments, cysteine of step (b) is at a concentrationin a range of from 0.2-5 mM (e.g., 0.2-4, 0.2-3, 0.5-5, 0.5-4, 0.5-2,0.5-1.5, 0.75-5, 0.75-3, or 0.75-1.25 mM, e.g., 1 mM). In someembodiments, zinc of step (b) is at a concentration in a range of from2-20 μM (e.g., 2-15, 5-20, 5-15, 7.5-20, 7.5-15, or 7.5-12.5 μM, e.g.,10 μM). In some embodiments, ALK inhibitor of step (b) is at aconcentration in a range of from 2-20 μM (e.g., 2-15, 5-20, 5-15,7.5-20, 7.5-15, or 7.5-12.5 μM, e.g., 10 μM). In some embodiments, BMPinhibitor LDN-193189 of step (b) is at a concentration in a range offrom 0.2-2 μM (e.g., 0.2-1.5, 0.2-1, 0.2-0.8, 0.3-2, 0.3-1.5, 0.3-1, or0.25-0.75 μM, e.g., 0.5 μM). In some embodiments, T3 thyroid hormone ofstep (b) is at a concentration in a range of from 0.2-5 μM (e.g., 0.2-4,0.2-3, 0.2-2, 0.5-5, 0.5-4, 0.5-3, 0.5-2, 0.5-1.5, 0.8-5, 0.8-4, 0.8-3,0.8-2, 0.8-1.5, or 0.8-1.2 μM, e.g., 1 μM). In some embodiments, gammasecretase inhibitor XX of step (b) is at a concentration in a range offrom 0.2-5 μM (e.g., 0.2-4, 0.2-3, 0.2-2, 0.5-5, 0.5-4, 0.5-3, 0.5-2,0.5-1.5, 0.8-5, 0.8-4, 0.8-3, 0.8-2, 0.8-1.5, or 0.8-1.2 μM, e.g., 1μM).

In some cases, the culture medium (e.g., DMEM) of step (b) comprises0.1-5 mM glutamine (e.g., 0.1-4, 0.1-3, 0.5-5, 0.5-4, 0.5-3, 1-5, 1-4,1-3, or 1.5-2.5 mM glutamine, e.g., 2 mM glutamine) or 0.05-2.5XGlutaMAX™ (e.g., 0.05-2X, 0.05-1.5X, 0.2-2.5X, 0.2-2X, 0.2-1.5X,0.5-2.5X, 0.5-2X, 0.5-1.5X, or 0.75-1.25X GlutaMAX, e.g., 1X GlutaMAX),and 0.1-5X non-essential amino acids (e.g., 0.1-4X, 0.1-3X, 0.5-5X,0.5-4X, 0.5-3X, 0.5-2X, 0.8-5X, 0.8-4X, 0.8-3X, 0.8-2X, 0.8X-1.5X, or0.8X-1.2X non-essential amino acids). In some cases, the culture medium(e.g., DMEM) of step (b) comprises 2 mM glutamine or 1X GlutaMAX™.

In some embodiments, vitamin C of step (c) is at a concentration in arange of from 10-2000 μM (e.g., 10-1500, 10-1250, 10-1000, 10-750,50-2000, 50-1500, 50-1250, 50-1000, 50-750, 100-2000, 100-1500,100-1250, 100-1000, 100-750, 200-2000, 200-1500, 200-1250, 200-1000,200-800, or 300-700 μM, e.g., 500 μM). In some embodiments, BayK-8644 ofstep (c) is at a concentration in a range of from 0.2-5 μM (e.g., 0.2-4,0.2-3, 0.5-5, 0.5-4, 0.5-3, 1-5, 1-4, 1-3, or 1.5-2.5 μM, e.g., 2 μM).

In addition, experiments disclosed herein have led to protocols forproducing pancreatic beta cells using the above-noted protocol, but atlower levels of oxygen, closer to the physiological situation in vivo.Also disclosed herein are data showing that the functional properties oftrue pancreatic beta cells generated under ambient oxygen levels (about20%) are vulnerable to reduction of in vitro O₂ to normal physiologicallevels (about 4-5% in vivo). This loss of functional properties uponreduction of ambient to physiological levels can be important for celltherapy and transplantation experiments. Cells cultured at ambientoxygen levels experience a loss of beta cell features upontransplantation into recipients where they immediately are exposed tophysiological oxygen levels. The data indicate that while some of thetransplanted pancreatic beta cells maintain a functional phenotype, manysuch cells lose essential functional features. This reduction infunctionality is connected to a loss of the differentiated phenotype andidentity of the pancreatic beta cells. Without intending to be bound byany particular theory, it is expected that the direct differentiation ofhES cells into pancreatic beta cells, wherein the hES cells have beenadapted to lower O₂ concentrations either during the pluripotent stateor after generation of pancreatic progenitor cells, represents a step indeveloping a successful cell therapy product for the treatment ofdiabetes by maintaining a functional pancreatic beta cell phenotype upontransplantation (FIG. 17).

Brief Description of Experimental Data

Experimental results described herein provide the firstproof-of-principle demonstration that functional definitive endodermcells, posterior foregut-like progenitor cells, and pancreaticprogenitor cells can be generated from fibroblast cells without goingthrough a pluripotent state. Compared with other approaches forgenerating such cells, the compositions and methods described herein canprovide sufficient numbers of functional progenitor cells fortransplantation and other applications.

Because pluripotent stem cells are not generated and the overall culturetime to obtain pancreatic cells is shortened, these methods are fasterand potentially safer than methods that utilize iPSCs. In addition tovalidating and extending the lineage-specific reprogramming paradigm togenerate cells of definitive endoderm and pancreatic lineages, severalnovel procedures illuminate to how lineage specification can beachieved, facilitated and maintained.

First, the methods and compositions include lineage specificationsignals (e.g., via TGFβ family members and WNT activators) at thebeginning of iPSC-factor-mediated epigenetic activation to significantlyimprove the efficiency of direct endodermal reprogramming Such a processmay transcriptionally program downstream lineage-specification signalsto specifically re-direct iPSC-factor-mediated epigenome remodelingbefore pluripotency is established. Thus, in addition to the benefits ofimproved reprogramming efficiency, providing lineage-specificationsignals early also ensures that pluripotent cells are not generated.

Prolonging the first step to 7 or 8 days increases the total colonynumber while dramatically decreasing the percentage of Sox17 and Foxa2double-positive colonies during expression of the pluripotency factors.The data provided herein indicate that temporary expression ofreprogramming factors is sufficient and even beneficial for achievingsuccessful conversion of cells to the endoderm lineage. Episomalexpression of pluripotency factors allows such transient expression,without insertion and potential mutation of chromosomal DNA. Suchtransient expression of reprogramming factors may be achieved withoutintegration of pluripotency expression cassettes into the genome ofstarting cells, to enhance the safety of this method (see, Kim, Kim etal. 2009; Zhou, Wu et al. 2009; Warren, Manos et al. 2010).

Secondly, the data described herein shows that while Pdx1 or Nkx6.1single-positive cells could be readily generated from definitiveendoderm cells, the cells so treated rarely co-express the other masterpancreatic specification genes and consequently fail to mature intoterminally differentiated, authentic pancreatic endocrine cells. Toovercome this challenge, unique combinations of small molecules wereidentified to generate Pdx1⁺/Nkx6.1⁺ cells. For example, a combinationof just four small molecules (retinoic acid, A83-01, LDE225 and pVc), inthe absence of any growth factors used in other protocols (such as FGF10and EGF), could not only effectively induce Pdx1⁺/Nkx6.1⁺ cells fromdefinitive endoderm cells, but also increase the percentage ofPdx1⁺/Nkx6.1⁺ cells produced by about 11-fold. The definitive endodermalprogenitor cells can rapidly be specified toward the posterior foregutstate (cPF), illustrating efficient transdifferentiation of thefibroblasts towards the endodermal germ layer. Moreover, the posteriorforegut-like progenitor cells can be greatly expanded without loss oftheir specific identity by culture in media that activates the WNTpathway and inhibits the TGFβ pathway while providing pro-proliferativegrowth factors EGF and bFGF. A somewhat unexpected finding was that notonly could foregut-like progenitor cells be greatly expanded, but onecould also expand pancreatic progenitor cells that were committedtowards a pancreatic phenotype. The expansion of pancreatic progenitorcells by at least two hundred million fold involved the addition of aselective set of factors, including growth factors (e.g., bFGF and EGF)as well as a TGFβ receptor inhibitor (e.g., A83-1).

Thirdly, experiments described herein show that influencing the earlysteps of lineage induction can significantly impact later specificationsteps. To date, efforts to influence lineage induction to a particularstage of development or differentiation have mostly focused on the stepimmediately preceding the particular stage, while earlier steps werelargely overlooked. For example, the search for small molecules thatimprove induction of Pdx1 pancreatic cells, MHC cardiac cells, or THdopamine neurons had always focused on using definitive endoderm cells,cardiac precursor cells, or neural precursor cells, respectively, forscreening. While this traditional approach is straightforward, thesuccessful procedures described herein demonstrate that enhancedattention at other differentiation stages can have significant benefits.To further improve conversion to pancreatic cells, new small moleculeswere tested and evaluated during the first two steps of reprogrammingtoward definitive endoderm cells using expression of Pdx1 and Nkx6.1 asindicators of differentiation into pancreatic progenitor cells. Forexample, when used in early cell-induction steps two small molecules,Bix-01294 and pVc, were found to substantially increase the number orpercentage of Pdx1⁺/Nkx6.1⁺ cells ultimately obtained. It should also benoted that treatment with Bix-01294 and pVc during the first two stepsof reprogramming did not affect the numbers of Sox17/Foxa2 positivecells obtained at about day 12 of the process. These results demonstratenew strategies for improving the outcome of later differentiation stagesin a step-wise cellular reprogramming and induction process by modifyingearlier steps, an approach that until now has been largely overlooked.

Novel combinations of small molecules that promote pancreatic beta-cellmaturation were also identified. For example, SB203580 and pVc stronglyand synergistically increased the number of insulin⁺/Pdx1⁺ cells. Notchinhibitor Compound-E, Vitamin C, and the Ca²⁺ channel agonist BayK-8644also improved the differentiation efficiency into pancreatic beta-likecells independently as well as in combination. Not only did thesescreenings reveal conditions that enable the reprogramming offibroblasts into endodermal and pancreatic progenitor cells that canmature into glucose-responsive insulin-secreting beta cells in vitro andin vivo, those newly identified small molecules for each step alsoprovide new chemical tools for further mechanistic studies.

The potential of the pancreatic progenitor cells generated as describedherein was demonstrated in vivo. The in vitro differentiated beta-likecells exhibited a mature, functional beta cell phenotype as judged bytheir ability to secrete insulin in response to physiological levels ofglucose as well as their co-expression of critical beta celltranscription factors. Compared with cells generated by previousconditions, the pancreatic progenitor cells generated by the advancedapproach described herein can much more efficiently generate all threepancreatic lineages, including functional insulin secreting beta likecells, and ameliorate hyperglycemia in vivo. These results demonstratethe therapeutic benefits of the methods and compositions describedherein.

Compositions

The invention also relates to compositions useful for generatingdefinitive endoderm cells, posterior foregut-like progenitor cells,pancreatic precursor cells, and/or mature pancreatic beta cells. Thecompositions can be formulated as cell culture media with one or more ofthe compounds and agents described herein at concentrations sufficientto generate definitive endoderm cells, posterior foregut-like progenitorcells, pancreatic precursor cells, and/or pancreatic beta cells.Alternatively, the compounds and agents described herein can be presentin concentrated or dry form within the compositions, so that thecomposition can conveniently be added to a cell culture medium togenerate a cell culture medium with an appropriate concentration of theselected compounds and/or agents for generating definitive endodermcells, posterior foregut-like progenitor cells, pancreatic precursorcells, and/or pancreatic beta cells.

Therefore, the invention relates to a first composition containing aTGFβ family member and a WNT activator. The first composition cancontain the TGFβ family member and the WNT activator in an effectiveamount that is sufficient to generate a first cell population comprisingdefinitive endoderm cells. The first composition can contain otherfactors. For example, the first composition can contain one or moregrowth factors (e.g., epidermal growth factor (EGF), basic fibroblastgrowth factor (bFGF), or a combination thereof). The first compositioncan also contain a G9a histone methyltransferase inhibitor,2-phospho-L-ascorbic acid, a histone deacetylase inhibitor, a histonedemethylase inhibitor, a DNA methyltransferase inhibitor, an adenosineagonist, or a combination thereof. The first composition can include oneor more of the agents or compounds listed above. Alternatively, thefirst compositions can contain two or more, or three or more, or four ormore, or five or more, or six or more, or seven or more of these agents.In some embodiments, the first composition has less than ten of theagents listed above, or less than nine of the agents listed above.

For example, Activin A (a TGFβ family member) can be present in thecompositions (useful e.g., for generating definitive endoderm cells) ata variety of concentrations, for example, at about 5 ng/ml to about 200ng/ml, or from about 10 ng/ml to about 175 ng/ml, or from about 15 ng/mlto about 150 ng/ml, or from about 20 ng/ml to about 150 ng/ml, or fromabout 25 ng/ml to about 125 ng/ml, or from about 30 ng/ml to about 100ng/ml, or from about 35 ng/ml to about 80 ng/ml, or from about 40 ng/mlto about 60 ng/ml, or about 50 ng/ml. Alternatively, the Activin A canbe present in concentrated form, for example, for addition to a cellculture medium so that the cell culture medium contains Activin A at adesired concentration. For example, a concentrated composition cancontain Activin A at a concentration that is about 10X, 50X, 100X, or1000X of the concentration at which it would be employed, for example,to generate definitive endoderm cells.

Lithium chloride and/or CHIR99021 (WNT activators) can be present in thecompositions (useful e.g., for generating definitive endoderm cells) ata variety of concentrations. For example, a WNT activator can beincluded at about 0.01 mM to about 10 mM, or from about 0.05 mM to about9 mM, or from about 0.1 mM to about 8 mM, or from about 0.2 mM to about7 mM, or from about 0.3 mM to about 6 mM, or from about 0.4 mM to about5 mM, or from about 0.5 mM to about 3 mM, or about 1 mM. CHIR99021 canbe present at 0.01 μM to about 10 μM, or from about 0.1 μM to about 8μM, or from about 1 μM to about 5 μM, or from about 2 μM to about 4 μM.Alternatively, the WNT activators can be present in concentrated form,for example, for addition to a cell culture medium so that the cellculture medium contains lithium chloride at a desired concentration. Forexample, a concentrated composition can contain lithium chloride and/orCHIR99021 at a concentration that is about 10X, 50X, 100X, or 1000X ofthe concentration at which it would be employed, for example, togenerate definitive endoderm cells.

Phospho-L-ascorbic acid or Vitamin C can be employed in the firstcomposition (useful e.g., for generating definitive endoderm cells) at avariety of concentrations, for example, at about 1 μM to about 1000 μM,or from about 20 μM to about 700 μM, or from about 50 μM to about 500μM, or from about 100 μM to about 400 μM, or from about 150 μM to about350 μM, or from about 200 μM to about 325 μM, or from about 250 μM toabout 310 μM, or about 280 μM. Alternatively, phospho-L-ascorbic acid orVitamin C can be present in concentrated form, for example, for additionto a cell culture medium so that the cell culture medium containsphospho-L-ascorbic acid at a desired concentration. For example, aconcentrated composition can contain phospho-L-ascorbic acid aconcentration that is about 10X, 50X, 100X, or 1000X of theconcentration at which it would be employed, for example, to generatedefinitive endoderm cells.

One or more G9a histone methyl-transferase inhibitors can be employed inthe first composition (useful e.g., for generating definitive endodermcells) at a variety of concentrations, for example, at about 0.01 μM toabout 10 μM, or from about 0.05 μM to about 9 μM, or from about 0.1 μMto about 8 μM, or from about 0.2 μM to about 7 μM, or from about 0.3 μMto about 6 μM, or from about 0.4 μM to about 5 μM, or from about 0.5 μMto about 3 μM, or about 1 μM. Alternatively, the G9a histonemethyl-transferase inhibitor can be present in concentrated form, forexample, for addition to a cell culture medium so that the cell culturemedium contains the G9a histone methyl-transferase inhibitor at adesired concentration. For example, a concentrated composition cancontain the G9a histone methyl-transferase inhibitor at a concentrationthat is about 10X, 50X, 100X, or 1000X of the concentration at which itwould be employed, for example, to generate definitive endoderm cells.

One or more histone deacetylase inhibitors can be employed in the firstcomposition (useful e.g., for generating definitive endoderm cells) at avariety of concentrations, for example, at about 1 micromolar to about20 millimolar, or about 10 micromolar to about 15 millimolar, or about25 micromolar to about 5 millimolar, or about 40 micromolar to about 1millimolar, or about 60 micromolar to about 0.5 millimolar, or about 0.1millimolar in a solution. For example, a concentrated composition cancontain one or more HDAC inhibitors at concentrations that are about10X, 50X, 100X, or 1000X of the concentration at which it would beemployed, for example, to generate definitive endoderm cells.

One or more histone demethylase inhibitors can be employed in the firstcomposition (useful e.g., for generating definitive endoderm cells) at avariety of concentrations, for example, about 0.01 micromolar to about20 micromolar, or about 0.05 micromolar to about 10 micromolar, or about0.1 micromolar to about 5 micromolar, or about 0.5 micromolar to about 3micromolar, or about 1 micromolar to about 3 micromolar, or about 1micromolar in a solution. For example, a concentrated composition cancontain one or more histone demethylase inhibitors at concentrationsthat are about 10X, 50X, 100X, or 1000X of the concentration at which itwould be employed, for example, to generate definitive endoderm cells.

One or more DNA methyltransferase inhibitors can be employed in thefirst composition (useful e.g., for generating definitive endodermcells) at a variety of concentrations, for example, at about 0.01micromolar to about 20 micromolar, or about 0.03 micromolar to about 10micromolar, or about 0.05 micromolar to about 5 micromolar, or about 0.1micromolar to about 2 micromolar, or about 0.2 micromolar to about 1micromolar, or about 0.5 micromolar in a solution. For example, aconcentrated composition can contain one or more DNA methyltransferaseinhibitors at concentrations that are about 10X, 50X, 100X, or 1000X ofthe concentration at which it would be employed, for example, togenerate definitive endoderm cells.

One or more adenosine agonists can be employed in the first composition(useful e.g., for generating definitive endoderm cells) at a variety ofconcentrations, for example, at about 0.01 micromolar to about 20micromolar, or about 0.03 micromolar to about 10 micromolar, or about0.05 micromolar to about 5 micromolar, or about 0.1 micromolar to about2 micromolar, or about 0.2 micromolar to about 1 micromolar, or about0.5 micromolar in a solution. For example, a concentrated compositioncan contain one or more adenosine agonists at concentrations that areabout 10X, 50X, 100X, or 1000X of the concentration at which it would beemployed, for example, to generate definitive endoderm cells.

The invention also relates to second compositions containing one or moreTGFβ receptor inhibitors, hedgehog pathway inhibitors, retinoic acidreceptor agonists, or combinations thereof. The second composition cancontain other factors. For example, the second composition can alsocontain one or more growth factors, WNT activators, vitamins (e.g.,vitamin C), Notch signaling inhibitors, BMP4 signaling inhibitors, orcombinations thereof. Any of such factors can be included in the secondcomposition in amounts sufficient to generate pancreatic progenitorcells from definitive endoderm cells.

One or more TGFβ receptor inhibitors can be employed in the secondcomposition (useful e.g., for generating pancreatic progenitor cells) ata variety of concentrations, for example, at about 0.01 μM to about 10μM, or from about 0.05 μM to about 9 μM, or from about 0.1 μM to about 8μM, or from about 0.2 μM to about 7 μM, or from about 0.3 μM to about 6μM, or from about 0.4 μM to about 5 μM, or from about 0.5 μM to about 3μM, or about 1 μM. For example, a concentrated composition can containone or more TGFβ receptor inhibitors at concentrations that are about10X, 50X, 100X, or 1000X of the concentration at which it would beemployed, for example, to generate pancreatic progenitor cells.

One or more hedgehog pathway inhibitors can be employed in the secondcomposition (useful e.g., for generating pancreatic progenitor cells) ata variety of concentrations, for example, at about 0.01 μM to about 10μM, or from about 0.05 μM to about 9 μM, or from about 0.1 μM to about 8μM, or from about 0.3 μM to about 7 μM, or from about 0.5 μM to about 6μM, or from about 0.75 μM to about 5 μM, or from about 1 μM to about 3μM, or about 2 μM. For example, a concentrated composition can containone or more hedgehog pathway inhibitors at concentrations that are about10X, 50X, 100X, or 1000X of the concentration at which it would beemployed, for example, to generate pancreatic progenitor cells.

One or more retinoic acid receptor agonists can be employed in thesecond composition (useful e.g., for generating pancreatic progenitorcells) at a variety of concentrations, for example, at about 0.01 μM toabout 10 μM, or from about 0.05 μM to about 9 μM, or from about 0.1 μMto about 8 μM, or from about 0.3 μM to about 7 μM, or from about 0.5 μMto about 6 μM, or from about 0.75 μM to about 5 μM, or from about 1 μMto about 3 μM, or about 2 μM. For example, a concentrated compositioncan contain one or more retinoic acid receptor agonists atconcentrations that are about 10X, 50X, 100X, or 1000X of theconcentration at which it would be employed, for example, to generatepancreatic progenitor cells.

One or more growth factors can be employed in the second composition(useful e.g., for generating pancreatic progenitor cells) at a varietyof concentrations, for example, at least about 1 ng/ml, or at leastabout 2 ng/ml, or at least about 3 ng/ml, or at least about 5 ng/ml, orabout 10 ng/ml. For example, a concentrated composition can contain oneor more growth factors at concentrations that are about 10X, 50X, 100X,or 1000X of the concentration at which it would be employed, forexample, to generate pancreatic progenitor cells.

One or more WNT activators can be employed in the second composition(useful e.g., for generating pancreatic progenitor cells) at a varietyof concentrations, for example, at about 0.01 micromolar to about 1millimolar in a solution, or about 0.1 micromolar to about 100micromolar in a solution, or about 0.5 micromolar to about 10 micromolarin a solution, or about 1 micromolar to about 5 micromolar in asolution. For example, a concentrated composition can contain one ormore WNT activators at concentrations that are about 10X, 50X, 100X, or1000X of the concentration at which it would be employed, for example,to generate pancreatic progenitor cells.

One or more vitamins (e.g., vitamin C or phospho-L-ascorbic acid) can beemployed in the second composition (useful e.g., for generatingpancreatic progenitor cells) at a variety of concentrations, forexample, at about 1 μM to about 1000 μM, or from about 20 μM to about700 μM, or from about 50 μM to about 500 μM, or from about 100 μM toabout 400 μM, or from about 150 μM to about 350 μM, or from about 200 μMto about 325 μM, or from about 250 μM to about 310 μM, or about 280 μM.For example, a concentrated composition can contain one or more vitaminsat concentrations that are about 10X, 50X, 100X, or 1000X of theconcentration at which it would be employed, for example, to generatepancreatic progenitor cells.

One or more Notch signaling inhibitors can be employed in the secondcomposition (useful e.g., for generating pancreatic progenitor cells) ata variety of concentrations, for example, at about 0.001 micromolar toabout 200 micromolar, or about 0.01 micromolar to about 100 micromolar,or about 0.05 micromolar to about 10 micromolar, or about 0.1 micromolarin a solution. For example, a concentrated composition can contain oneor more Notch signaling inhibitors at concentrations that are about 10X,50X, 100X, or 1000X of the concentration at which it would be employed,for example, to generate pancreatic progenitor cells.

One or more BMP4 signaling inhibitors can be employed in the secondcomposition (useful e.g., for generating pancreatic progenitor cells) ata variety of concentrations, for example, at about 0.001 micromolar toabout 200 micromolar, or about 0.01 micromolar to about 100 micromolar,or about 0.05 micromolar to about 10 micromolar, or about 0.1 micromolarin a solution. BMP4 signaling inhibitors at concentrations that areabout 10X, 50X, 100X, or 1000X of the concentration at which it would beemployed, for example, to generate pancreatic progenitor cells.

The invention also relates to third compositions containing one or moreTGFβ receptor inhibitors, Notch inhibitors, polyADP ribose synthetaseinhibitors, or a combination thereof useful for generating pancreaticbeta cells from pancreatic progenitor cells. The third composition cancontain other factors. For example, the third composition can alsocontain one or more glucagon-like peptide-1 agonists, vitamins, p38mitogen-activated protein (MAP) kinase inhibitors, basement membraneproteins, adenylyl cyclase activators, glucocorticoid receptor agonists,Ca²⁺ channel agonists, or combinations thereof.

One or more TGFβ receptor inhibitors can be employed in the thirdcomposition (useful e.g., for generating pancreatic beta cells) at avariety of concentrations, for example, at about 0.01 μM to about 10 μM,or from about 0.05 μM to about 9 μM, or from about 0.1 μM to about 8 μM,or from about 0.2 μM to about 5 μM, or from about 0.3 μM to about 3 μM,or from about 0.3 μM to about 2 μM, or from about 0.4 μM to about 1 μM,or about 0.5 μM. For example, a concentrated composition can contain oneor more TGFβ receptor inhibitors at concentrations that are about 10X,50X, 100X, or 1000X of the concentration at which it would be employed,for example, to generate pancreatic beta cells.

One or more Notch inhibitors can be employed in the third composition(useful e.g., for generating pancreatic beta cells) at a variety ofconcentrations, for example, at about 0.001 micromolar to about 200micromolar, or about 0.01 micromolar to about 100 micromolar, or about0.05 micromolar to about 10 micromolar, or about 0.1 micromolar in asolution. For example, a concentrated composition can contain one ormore Notch inhibitors at concentrations that are about 10X, 50X, 100X,or 1000X of the concentration at which it would be employed, forexample, to generate pancreatic beta cells.

One or more polyADP ribose synthetase inhibitors can be employed in thethird composition (useful e.g., for generating pancreatic beta cells) ata variety of concentrations, for example, at about 0.1 millimolar toabout 100 millimolar, or about 1 millimolar to about 50 millimolar, orabout 3 millimolar to about 30 millimolar, or about 5 micromolar toabout 15 millimolar, or about 10 millimolar in a solution. For example,a concentrated composition can contain one or more polyADP ribosesynthetase inhibitors at concentrations that are about 10X, 50X, 100X,or 1000X of the concentration at which it would be employed, forexample, to generate pancreatic beta cells.

One or more glucagon-like peptide-1 agonists can be employed in thethird composition (useful e.g., for generating pancreatic beta cells) ata variety of concentrations, for example, at about 5 ng/ml to about 200ng/ml, or from about 10 ng/ml to about 175 ng/ml, or from about 15 ng/mlto about 150 ng/ml, or from about 20 ng/ml to about 150 ng/ml, or fromabout 25 ng/ml to about 125 ng/ml, or from about 30 ng/ml to about 100ng/ml, or from about 35 ng/ml to about 80 ng/ml, or from about 40 ng/mlto about 60 ng/ml, or about 50 ng/ml. For example, a concentratedcomposition can contain one or more glucagon-like peptide-1 agonists atconcentrations that are about 10X, 50X, 100X, or 1000X of theconcentration at which it would be employed, for example, to generatepancreatic beta cells.

One or more vitamins (e.g., vitamin C and/or phospho L-ascorbic acid)can be employed in the third composition (useful e.g., for generatingpancreatic beta cells) at a variety of concentrations, for example, atabout 1 μM to about 100 μM, or from about 10 μM to about 80 μM, or fromabout 25 μM to about 75 μM, or from about 30 μM to about 70 μM, or fromabout 35 μM to about 65 μM, or from about 40 μM to about 60 μM, or fromabout 45 μM to about 55 μM, or about 50 μM. For example, a concentratedcomposition can contain one or more vitamins (e.g., vitamin C and/orphospho L-ascorbic acid) at concentrations that are about 10X, 50X,100X, or 1000X of the concentration at which it would be employed, forexample, to generate pancreatic beta cells.

One or more p38 mitogen-activated protein (MAP) kinase inhibitors can beemployed in the third composition (useful e.g., for generatingpancreatic beta cells) at a variety of concentrations, for example, atabout 0.1 μM to about 500 μM, or from about 0.5 μM to about 400 μM, orfrom about 1 μM to about 200 μM, or from about 2 μM to about 100 μM, orfrom about 2.5 μM to about 50 μM, or from about 3 μM to about 25 μM, orfrom about 4 μM to about 10 μM, or about 5 μM. For example, aconcentrated composition can contain one or more p38 mitogen-activatedprotein (MAP) kinase inhibitors at concentrations that are about 10X,50X, 100X, or 1000X of the concentration at which it would be employed,for example, to generate pancreatic beta cells.

One or more basement membrane proteins can be employed in the thirdcomposition (useful e.g., for generating pancreatic beta cells) at avariety of concentrations, for example, at about 0.05 μM to about 200μM, or from about 0.1 μM to about 100 μM, or from about 0.2 μM to about50 μM, or from about 0.3 μM to about 25 μM, or from about 0.5 μM toabout 10 μM, or from about 1 μM to about 5 μM, or from about 1.5 μM toabout 4 μM, or about 2 μM. For example, a concentrated composition cancontain one or more basement membrane proteins at concentrations thatare about 10X, 50X, 100X, or 1000X of the concentration at which itwould be employed, for example, to generate pancreatic beta cells.

One or more adenylyl cyclase activators can be employed in the thirdcomposition (useful e.g., for generating pancreatic beta cells) at avariety of concentrations, for example, at about at about 0.1 μM toabout 500 μM, or from about 0.5 μM to about 400 μM, or from about 1 μMto about 200 μM, or from about 2 μM to about 100 μM, or from about 2.5μM to about 50 μM, or from about 3 μM to about 25 μM, or from about 5 μMto about 15 μM, or about 10 μM. For example, a concentrated compositioncan contain one or more adenylyl cyclase activators at concentrationsthat are about 10X, 50X, 100X, or 1000X of the concentration at which itwould be employed, for example, to generate pancreatic beta cells.

One or more glucocorticoid receptor agonists can be employed in thethird composition (useful e.g., for generating pancreatic beta cells) ata variety of concentrations, for example, at about 0.1 μM to about 500μM, or from about 0.5 μM to about 400 μM, or from about 1 μM to about200 μM, or from about 2 μM to about 100 μM, or from about 2.5 μM toabout 50 μM, or from about 3 μM to about 25 μM, or from about 5 μM toabout 15 μM, or about 10 μM. For example, a concentrated composition cancontain one or more glucocorticoid receptor agonists at concentrationsthat are about 10X, 50X, 100X, or 1000X of the concentration at which itwould be employed, for example, to generate pancreatic beta cells.

One or more Ca²⁺ channel agonists can be employed in the thirdcomposition (useful e.g., for generating pancreatic beta cells) at avariety of concentrations, for example, at about 0.05 μM to about 200μM, or from about 0.1 μM to about 100 μM, or from about 0.2 μM to about50 μM, or from about 0.3 μM to about 25 μM, or from about 0.5 μM toabout 10 μM, or from about 1 μM to about 5 μM, or from about 1.5 μM toabout 4 μM, or about 2 μM. For example, a concentrated composition cancontain one or more Ca²⁺ channel agonists at concentrations that areabout 10X, 50X, 100X, or 1000X of the concentration at which it would beemployed, for example, to generate pancreatic beta cells.

Other compositions useful for converting pancreatic endodermalprogenitor cells to pancreatic beta-like cells include those employedduring protocol 2. For example, one example of a protocol 2 compositionis a composition that includes heparin, zinc salts, a TGF-β inhibitor(e.g., Alk5 inhibitor), a BMP4 signaling inhibitor (e.g., LDN-193189), aT3 thyroid hormone, a Notch Signaling Inhibitor (e.g., Compound E), aCa²⁺ channel agonist (e.g. BayK-8644), vitamin C, and combinationsthereof. Another example of a protocol 2 composition is one thatincludes heparin zinc salts, a TGF-β inhibitor (e.g., Alk5 inhibitor), aBMP4 signaling inhibitor (e.g., LDN-193189), a T3 thyroid hormone,cysteine, an anti-oxidant (e.g., vitamin E or a derivative thereof suchas Trolox), an Axl kinase inhibitor (e.g., R428), a Ca²⁺ channel agonist(e.g. BayK-8644), vitamin C, and combinations thereof to generate asecond population of pancreatic beta-like cells. The protocol 2compositions can contain these ingredients in amounts and atconcentrations that are described herein.

The compositions can be cell culture media, or they can includecomponents of a culture medium.

Kits

Also provided are kits for generating definitive endoderm cells and/orpancreatic precursor cells and/or pancreatic cells. The kits can containany of the compositions described herein and instructions for using thecompositions for generating definitive endoderm cells and/or pancreaticprecursor cells and/or pancreatic cells. Each of the compositions can beseparately packaged. Each composition can contain any of the compoundsor proteins described herein at a concentration that is convenient foraddition to a culture of cells. For example, compositions can beconcentrated to about 10X, 50X, 100X, or 1000X of the concentration atwhich it would be employed to generate definitive endoderm cells and/orpancreatic precursor cells and/or posterior foregut-like progenitorcells and/or pancreatic beta cells. The instructions can provideguidance for appropriate addition (dilution) of the compositions into acell culture, and/or guidance on other culture conditions (e.g.,appropriate cell culture media, an appropriate duration of exposure tothe compositions, etc.). The instructions can also provide guidance onthe selection of cells for generating definitive endoderm cells and/orpancreatic precursor cells and/or posterior foregut-like progenitorcells and/or pancreatic cells. In addition, the instructions can provideinformation for testing and/or recognition of the generated definitiveendoderm cells and/or pancreatic precursor cells and/or posteriorforegut-like progenitor cells and/or pancreatic cells.

The kits can also provide components and instructions for administeringdefinitive endoderm cells and/or pancreatic precursor cells and/orposterior foregut-like progenitor cells and/or pancreatic beta cells tomammalian subjects. The instructions can provide guidance on the numbersand the type(s) (phenotype) of cells to be administered. Theinstructions can also provide instructions for administration ofdefinitive endoderm cells and/or pancreatic precursor cells and/orposterior foregut-like progenitor cells and/or pancreatic beta cells bysurgical implantation or by infusion. For example, the kits can providediluents, pharmaceutically acceptable carriers, scalpels, syringes,catheters, bandages, antiseptics, and the like to permit administrationof cells.

Mixtures

The definitive endoderm cells and/or posterior foregut-like progenitorcells and/or pancreatic precursor cells and/or pancreatic cells can bepresent in any of the foregoing compositions. The definitive endodermcells and/or posterior foregut-like progenitor cells and/or pancreaticprecursor cells and/or pancreatic cells can also be present in atherapeutically acceptable carrier such as saline, phosphate bufferedsaline, or other aqueous carrier. Such a combination of the compositionsdescribed herein, or a therapeutically acceptable carrier, withdefinitive endoderm cells and/or posterior foregut-like progenitor cellsand/or pancreatic precursor cells and/or pancreatic cells is called amixture.

The mixtures can contain about 1 to about 10¹⁰ definitive endoderm cellsand/or posterior foregut-like progenitor cells and/or pancreaticprecursor cells and/or pancreatic (e.g., beta) cells.

The definitive endoderm cells and/or posterior foregut-like progenitorcells and/or pancreatic precursor cells and/or pancreatic cellsgenerated as described herein can be isolated, separated, or purifiedfrom the composition, mixture or media in which they are generated. Thedefinitive endoderm cells and/or posterior foregut-like progenitor cellsand/or pancreatic precursor cells and/or pancreatic cells generated asdescribed herein can be enriched or cultured to increase the proportionor numbers of desired cells in the population. Any such isolate,separation, purification, enrichment or culture is a mixture of theinvention.

An isolating step can include providing the cells in the cell culturewith a reagent which binds to a marker expressed in the desired celltype (e.g., definitive endoderm cells, pancreatic precursor cells and/orpancreatic cells) but which is not substantially expressed in othercells present in the cell culture. The reagent-bound cells can beseparated from the non-reagent-bound cells by numerous methods. Forexample, an antibody against a marker that is selectively present on thedesired cells can be provided to cells in a cell culture. Antibody-boundcells can then be separated from other cells in the culture by, forexample, fluorescent activated cell sorting (FACS), binding the antibodyto a solid support or isolating appropriately tagged antibody in amagnetic field. In some embodiments, the antibody is released from thecells after the separation process.

As an alternative means of separation, at least some of the desiredcells can be separated from at least some of the other cells in theculture by specifically fluorescently labeling the desired cells inculture and then separating the labeled cells from the unlabeled cellsby FACS.

An enriched cell population of definitive endoderm cells, posteriorforegut-like progenitor cells, pancreatic progenitor cells, and/orpancreatic beta cells produced, for example, by an isolating step can besubstantially free of cells other than definitive endoderm cells,posterior foregut-like progenitor cells, pancreatic progenitor cells,and/or pancreatic beta cells. In other embodiments, the enriched cellpopulations can have at least about 50% to at least about 100%definitive endoderm cells, posterior foregut-like progenitor cells,pancreatic progenitor cells, and/or pancreatic beta cells. In stillother embodiments, the enriched cell populations comprise from at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 96%, at least about 97%, at leastabout 99%, or at least about 100% definitive endoderm cells, posteriorforegut-like progenitor cells, pancreatic progenitor cells, and/orpancreatic beta cells.

In some instances, the definitive endoderm cells, posterior foregut-likeprogenitor cells, pancreatic progenitor cells, and/or pancreatic betacells are expanded, for example, by culturing the cells under conditionsthat permit cell division. For example, some embodiments include aculturing step that comprises plating a cell population on a surfacesuch as a culture plate. In some embodiments, the cells are plated on asurface coated with a protein, poly-amino acid or carbohydrate (e.g.,collagen, fibronectin, polylysine, poly-ornithine).

In other embodiments, the culturing step comprises incubating the cellpopulation or portion thereof in an expansion medium comprising about 2%(v/v) serum. In some embodiments, the serum concentration can range fromabout 0% (v/v) to about 20% (v/v). For example, in some methodsdescribed herein, the serum concentration of the medium can be about0.05% (v/v), about 0.1% (v/v), about 0.2% (v/v), about 0.3% (v/v), about0.4% (v/v), about 0.5% (v/v), about 0.6% (v/v), about 0.7% (v/v), about0.8% (v/v), about 0.9% (v/v), about 1% (v/v), about 2% (v/v), about 3%(v/v), about 4% (v/v), about 5% (v/v), about 6% (v/v), about 7% (v/v),about 8% (v/v), about 9% (v/v), about 10% (v/v), about 15% (v/v) orabout 20% (v/v). In some embodiments, serum replacement is included inthe medium, and no serum is employed.

Using the methods described herein, cell populations or cell culturescan be enriched in definitive endoderm, posterior foregut-likeprogenitor, pancreatic progenitor, and/or pancreatic beta cell contentby at least about 2-fold to about 1000-fold as compared to cellpopulations or cell cultures produced by the methods and compositionsdescribed herein. In some embodiments, definitive endoderm, posteriorforegut-like progenitor, pancreatic progenitor, and/or pancreatic betacells can be enriched by at least about 5-fold to about 500-fold ascompared to cell populations or cell cultures produced by the methodsand compositions described herein. In other embodiments, definitiveendoderm, posterior foregut-like progenitor, pancreatic progenitor,and/or pancreatic beta cells can be enriched from at least about 10- toabout 200-fold as compared to cell populations or cell cultures producedby the methods and compositions described herein. In still otherembodiments, definitive endoderm, posterior foregut-like progenitor,pancreatic progenitor, and/or pancreatic beta cells can be enriched fromat least about 20- to about 100-fold as compared to cell populations orcell cultures produced by the methods and compositions described herein.In yet other embodiments, definitive endoderm, posterior foregut-likeprogenitor, pancreatic progenitor, and/or pancreatic beta cells can beenriched from at least about 40- to about 80-fold as compared to cellpopulations or cell cultures produced by the methods and compositionsdescribed herein. In certain embodiments, definitive endoderm, posteriorforegut-like progenitor, pancreatic progenitor, and/or pancreatic betacells can be enriched from at least about 2- to about 20-fold ascompared to cell populations or cell cultures produced by the methodsand compositions described herein.

Some embodiments described herein relate to cell cultures or cellpopulations comprising from at least about 5% definitive endoderm,posterior foregut-like progenitor, pancreatic progenitor, and/orpancreatic beta cells to at least about 95% definitive endoderm,pancreatic progenitor, and/or pancreatic beta cells. In some embodimentsthe cell cultures or cell populations comprise mammalian cells. Inpreferred embodiments, the cell cultures or cell populations comprisehuman cells. For example, certain specific embodiments relate to cellcultures comprising human cells, wherein from at least about 5% to atleast about 95% of the human cells are definitive endoderm, posteriorforegut-like progenitor, pancreatic progenitor, and/or pancreatic betacells. Other embodiments relate to cell cultures comprising human cells,wherein at least about 5%, at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90% or greater than 90% of the human cells are definitiveendoderm, posterior foregut-like progenitor, pancreatic progenitor,and/or pancreatic beta cells. In embodiments where the cell cultures orcell populations comprise human feeder cells, the above percentages arecalculated without respect to the human feeder cells in the cellcultures or cell populations.

Therapy

Also described herein is a method for treating a patient suffering from,or at risk of developing, diabetes. The diabetes can, for example, betype 1, type 2, or type 1.5 diabetes. This method involves obtainingdefinitive endoderm, pancreatic progenitor, and/or pancreatic beta cellsas described herein, and administering or implanting the cells into amammalian subject.

The definitive endoderm, pancreatic progenitor, and/or pancreatic betacells can be implanted as dispersed cells or formed into clusters.Alternatively, the definitive endoderm, pancreatic progenitor, and/orpancreatic beta cells can be infused into the subject, for example, viaa hepatic portal vein. Alternatively, cells may be provided inbiocompatible degradable polymeric supports, porous non-degradabledevices or encapsulated to protect from host immune response. Cells maybe implanted into an appropriate site in a subject. The implantationsites include, for example, the liver, natural pancreas, renalsubcapsular space, omentum, peritoneum, subserosal space, intestine,stomach, or a subcutaneous pocket.

The amount of cells used in implantation depends on a number of variousfactors including the subject's condition and response to the therapy,and can be determined by one skilled in the art. For example, the numberof cells administered can range from about 1000 to about 10¹⁰.

In one aspect, this invention provides a method for treating a patientsuffering from, or at risk of developing diabetes. This method involvesculturing a selected cell population, differentiating or redirecting thecultured cells in vitro into a definitive endoderm lineage to generate afirst cell population containing definitive endoderm cells,differentiating the first cell population into a second populationcontaining pancreatic progenitor cells and administering the secondpopulation of cells to a subject. In some instances pancreaticprogenitor cells are enriched within the second population or purifiedfrom the second population of cells to generate a third population ofcells that is substantially free of non-pancreatic cells.

The cells to be administered can be incorporated into athree-dimensional support. The cells can be maintained in vitro on thissupport prior to implantation into the subject. Alternatively, thesupport containing the cells can be directly implanted in the subjectwithout additional in vitro culturing. The support can optionally beincorporated with at least one pharmaceutical agent that facilitates thesurvival and function of the transplanted cells.

Support materials suitable for use include tissue templates, conduits,barriers, and reservoirs useful for tissue repair. In particular,synthetic and natural materials in the form of foams, sponges, gels,hydrogels, textiles, and nonwoven structures, which have been used invitro and in vivo to reconstruct or regenerate biological tissue, aswell as to deliver chemotactic agents for inducing tissue growth, aresuitable for use in practicing the methods described herein. See, forexample, the materials disclosed in U.S. Pat. No. 5,770,417, U.S. Pat.No. 6,022,743, U.S. Pat. No. 5,567,612, U.S. Pat. No. 5,759,830, U.S.Pat. No. 6,626,950, U.S. Pat. No. 6,534,084, U.S. Pat. No. 6,306,424,U.S. Pat. No. 6,365,149, U.S. Pat. No. 6,599,323, U.S. Pat. No.6,656,488, U.S. Published Application 2004/0062753 A1, U.S. Pat. No.4,557,264 and U.S. Pat. No. 6,333,029, each of which is specificallyincorporated by reference herein in its entirety.

The mammalian subject can be a human patient, a domestic animal, or alaboratory animal.

Definitions

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a compound,” “a cell,” “anucleic acid” or “a polypeptide” includes a plurality of such compounds,cells, nucleic acids or polypeptides (for example, a solution of cells,nucleic acids or polypeptides, a suspension of cells, or a series ofcompound, cell, nucleic acid or polypeptide preparations), and so forth.

The terms “diabetes” and “diabetes mellitus” are used interchangeablyherein. The World Health Organization indicates that diabetes involves afasting plasma glucose concentration of 7.0 mmol/1 (126 mg/dl) and abovefor Diabetes Mellitus (whole blood 6.1 mmol/1 or 110 mg/dl), or 2-hourglucose level 11.1 mmol/L or higher (200 mg/dL or higher). Other valuessuggestive of or indicating high risk for Diabetes Mellitus includeelevated arterial pressure 140/90 mm Hg or higher; elevated plasmatriglycerides (1.7 mmol/L; 150 mg/dL) and/or low HDL-cholesterol (lessthan 0.9 mmol/L, 35 mg/dl for men; less than 1.0 mmol/L, 39 mg/dLwomen); central obesity (males: waist to hip ratio higher than 0.90;females: waist to hip ratio higher than 0.85) and/or body mass indexexceeding 30 kg/m2; microalbuminuria, where the urinary albuminexcretion rate 20 μg/min or higher, or albumin to creatinine ratio 30mg/g or higher). The term diabetes encompasses all forms of diabetes,including Type I, Type II and Type 1.5.

As used herein, the term “treating” and “treatment” refers toadministering to a subject an effective amount of a composition so thatthe recipient has a reduction in at least one symptom of the disease oran improvement in the disease, for example, beneficial or desiredclinical results. For purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation of one ormore symptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. Treating canrefer to prolonging survival as compared to expected survival if notreceiving treatment. Thus, one of skill in the art realizes that atreatment may improve the disease condition, but may not be a completecure for the disease. The term “treatment” includes prophylaxis.Treatment is “effective” if the progression of a disease is reduced orhalted. “Treatment” can also mean prolonging survival as compared toexpected survival if not receiving treatment. Those in need of treatmentinclude those already diagnosed with a condition (e.g., diabetes), aswell as those likely to develop a condition due to geneticsusceptibility or other factors such as weight, diet, and health.

The term “episomal” refers to the extra-chromosomal state of anexpression cassette, plasmid or vector in a cell. Episomal expressioncassettes, plasmids or vectors are nucleic acid molecules that are notpart of the chromosomal DNA and replicate independently thereof.

An “Oct polypeptide” refers to any of the naturally-occurring members ofOctamer family of transcription factors, or variants thereof thatmaintain transcription factor activity, similar (within at least 50%,80%, or 90% activity) compared to the closest related naturallyoccurring family member, or polypeptides comprising at least theDNA-binding domain of the naturally occurring family member, and canfurther comprise a transcriptional activation domain. Exemplary Octpolypeptides include, Oct-1, Oct-2, Oct-3/4, Oct-6, Oct-7, Oct-8, Oct-9,and Oct-11. e.g., Oct3/4 (referred to herein as “Oct4”) contains the POUdomain, a 150 amino acid sequence conserved among Pit-1, Oct-1, Oct-2,and uric-86. See, Ryan, A. K. & Rosenfeld, M. G. Genes Dev. 11,1207-1225 (1997). In some embodiments, variants have at least 85%, 90%,or 95% amino acid sequence identity across their whole sequence comparedto a naturally occurring Oct polypeptide family member such as to thoselisted above or such as listed in Genbank accession number NP-002692.2(human Oct4) or NP-038661.1 (mouse Oct4). Oct polypeptides (e.g.,Oct3/4) can be from human, mouse, rat, bovine, porcine, or otheranimals. Generally, the same species of protein will be used with thespecies of cells being manipulated. The Oct polypeptide(s) can be apluripotency factor.

A “Klf polypeptide” refers to any of the naturally-occurring members ofthe family of Krüppel-like factors (Klfs), zinc-finger proteins thatcontain amino acid sequences similar to those of the Drosophilaembryonic pattern regulator Krüppel, or variants of thenaturally-occurring members that maintain transcription factor activitysimilar (within at least 50%, 80%, or 90% activity) compared to theclosest related naturally occurring family member, or polypeptidescomprising at least the DNA-binding domain of the naturally occurringfamily member, and can further comprise a transcriptional activationdomain. See, Dang, D. T., Pevsner, J. & Yang, V. W. Cell Biol. 32,1103-1121 (2000). Exemplary Klf family members include, Klf1, Klf2,Klf3, Klf-4, Klf5, Klf6, Klf7, Klf8, Klf9, Klf10, Klf11, Klf12, Klf13,Klf14, Klf15, Klf16, and Klf17. Klf2 and Klf-4 were found to be factorscapable of generating iPS cells in mice, and related genes Klf1 and Klf5did as well, although with reduced efficiency. See, Nakagawa, et al.,Nature Biotechnology 26:101-106 (2007). In some embodiments, variantshave at least 85%, 90%, or 95% amino acid sequence identity across theirwhole sequence compared to a naturally occurring Klf polypeptide familymember such as to those listed above or such as listed in Genbankaccession number CAX16088 (mouse Klf4) or CAX14962 (human Klf4). Klfpolypeptides (e.g., Klf1, Klf4, and Klf5) can be from human, mouse, rat,bovine, porcine, or other animals. Generally, the same species ofprotein will be used with the species of cells being manipulated. To theextent a Klf polypeptide is described herein, it can be replaced with anestrogen-related receptor beta (Essrb) polypeptide. Thus, it is intendedthat for each Klf polypeptide embodiment described herein, acorresponding embodiment using Essrb in the place of a Klf4 polypeptideis equally described. The Klf polypeptide(s) can be a pluripotencyfactor.

A “Myc polypeptide” refers any of the naturally-occurring members of theMyc family (see, e.g., Adhikary, S. & Eilers, M. Nat. Rev. Mol. CellBiol. 6:635-645 (2005)), or variants thereof that maintain transcriptionfactor activity similar (within at least 50%, 80%, or 90% activity)compared to the closest related naturally occurring family member, orpolypeptides comprising at least the DNA-binding domain of the naturallyoccurring family member, and can further comprise a transcriptionalactivation domain. Exemplary Myc polypeptides include, e.g., c-Myc,N-Myc and L-Myc. In some embodiments, variants have at least 85%, 90%,or 95% amino acid sequence identity across their whole sequence comparedto a naturally occurring Myc polypeptide family member, such as to thoselisted above or such as listed in Genbank accession number CAA25015(human Myc). Myc polypeptides (e.g., c-Myc) can be from human, mouse,rat, bovine, porcine, or other animals. Generally, the same species ofprotein will be used with the species of cells being manipulated. TheMyc polypeptide(s) can be a pluripotency factor.

A “Sox polypeptide” refers to any of the naturally-occurring members ofthe SRY-related HMG-box (Sox) transcription factors, characterized bythe presence of the high-mobility group (HMG) domain, or variantsthereof that maintain transcription factor activity similar (within atleast 50%, 80%, or 90% activity) compared to the closest relatednaturally occurring family member, or polypeptides comprising at leastthe DNA-binding domain of the naturally occurring family member, and canfurther comprise a transcriptional activation domain. See, e.g., Dang,D. T., et al., Int. J. Biochem Cell Biol. 32:1103-1121 (2000). ExemplarySox polypeptides include, e.g., Sox1, Sox-2, Sox3, Sox4, Sox5, Sox6,Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17,Sox18, Sox-21, and Sox30. Sox1 has been shown to yield iPS cells with asimilar efficiency as Sox2, and genes Sox3, Sox15, and Sox18 have alsobeen shown to generate iPS cells, although with somewhat less efficiencythan Sox2. See, Nakagawa, et al., Nature Biotechnology 26:101-106(2007). In some embodiments, variants have at least 85%, 90%, or 95%amino acid sequence identity across their whole sequence compared to anaturally occurring Sox polypeptide family member such as to thoselisted above or such as listed in Genbank accession number CAA83435(human Sox2). Sox polypeptides (e.g., Sox1, Sox2, Sox3, Sox15, or Sox18)can be from human, mouse, rat, bovine, porcine, or other animalsGenerally, the same species of protein will be used with the species ofcells being manipulated. The Sox polypeptide(s) can be a pluripotencyfactor.

“H3K9” refers to histone H3 lysine 9. H3K9 modifications associated withgene activity include H3K9 acetylation and H3K9 modifications associatedwith heterochromatin, include H3K9 di-methylation or tri-methylation.See, e.g., Kubicek, et al., Mol. Cell 473-481 (2007).

A “p53 inhibitor” refers to a molecule that reduces p53 activity andexpression. Such a p53 inhibitor can reduce the activity of a p53protein, the translation of the p53 protein, the expression of a p53gene, or a combination thereof. Examples of a p53 inhibitor include, butare not limited to nucleic acids, proteins, dominant negative proteins,peptides, oligosaccharides, polysaccharides, lipids, phospholipids,glycolipids, monomers, polymers, small molecules and organic compounds.The p53 inhibitor can be a polynucleotide such as a nucleic acid segmentoperably linked to a promoter. In some embodiments, the p53 inhibitor isa short hairpin RNA. In other embodiments, the p53 inhibitor is a smallinterfering RNA. The p53 inhibitor may be a protein. In someembodiments, the p53 inhibitor is a dominant negative protein.Information on such p53 inhibitors is available, for example, in U.S.Pat. No. 8,530,238 by Yamanka et al.; Yu et al., Human inducedpluripotent stem cells free of vector and transgene sequences, Science324(5928): 797-801 (2009); United States Patent Application 20120076762,and Okita et al., A more efficient method to generate integration-freehuman iPS cells, Nature Methods 8: 409-412 (2011), the contents of whichare specifically incorporated herein by reference in their entireties.The “p53 shRNA” can, for example, be a shRNA against p53 with thesequence described in U.S. Pat. No. 8,530,238 by Yamanka et al. or Honget al. (Nature. 460: 1132-1135 (2009)), both of which are incorporatedherein by reference in their entirety.

“NANOG” as referred to herein includes any of the naturally-occurringforms of the Nanog transcription factor, or variants thereof thatmaintain NANOG transcription factor activity (e.g. with at least 50%,80%, 90% or 100% activity of the activity of natural NANOG). In someembodiments, variants have at least 90% or 95% amino acid sequenceidentity across their whole sequence compared to the naturally occurringNANOG polypeptide. For example, the NANOG protein can have the sequenceidentified by the NCBI reference gi:153945816.

The terms “transfection” or “transfected” are defined by a process ofintroducing nucleic acid molecules into a cell by non-viral andviral-based methods. The nucleic acid molecules may be gene sequencesencoding complete proteins or functional portions thereof.

The term “pluripotent” or “pluripotency” refers to cells with theability to give rise to progeny cells that can undergo differentiation,under the appropriate conditions, into cell types that collectivelydemonstrate characteristics associated with cell lineages from all ofthe three germinal layers (endoderm, mesoderm, and ectoderm).Pluripotent stem cells can contribute to all embryonic derived tissuesof a prenatal, postnatal or adult animal A standard art-accepted test,such as the ability to form a teratoma in 8-12 week old SCID mice, canbe used to establish the pluripotency of a cell population, howeveridentification of various pluripotent stem cell characteristics can alsobe used to detect pluripotent cells.

“Pluripotent stem cell characteristics” refer to characteristics of acell that distinguish pluripotent stem cells from other cells. Theability to give rise to progeny that can undergo differentiation, underthe appropriate conditions, into cell types that collectivelydemonstrate characteristics associated with cell lineages from all ofthe three germinal layers (endoderm, mesoderm, and ectoderm) is apluripotent stem cell characteristic. Expression or non-expression ofcertain combinations of molecular markers are also pluripotent stem cellcharacteristics. For example, human pluripotent stem cells express atleast some, and in some embodiments, all of the markers from thefollowing non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81,TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog. Cellmorphologies associated with pluripotent stem cells are also pluripotentstem cell characteristics.

As used herein, “multipotent” or “multipotent cell” refers to a celltype that can give rise to a limited number of other particular celltypes.

As used herein, “non-pluripotent cells” refer to mammalian cells thatare not pluripotent cells. Examples of such cells include differentiatedcells as well as progenitor cells. Examples of differentiated cellsinclude, but are not limited to, cells from a tissue selected from bonemarrow, skin, skeletal muscle, fat tissue and peripheral blood.Exemplary cell types include, but are not limited to, fibroblasts,epidermal cells, lymphocytes, hepatocytes, myoblasts, neurons,osteoblasts, osteoclasts, and T-cells.

Where an individual is to be treated with definitive endoderm cells,pancreatic progenitor cells, and/or pancreatic beta cells, theindividual's own non-pluripotent cells can be used to generatedefinitive endoderm cells, pancreatic progenitor cells, and/orpancreatic beta cells according to the methods of the invention.

Mammalian cells can be from humans or non-human mammals. Exemplarynon-human mammals include, but are not limited to, mice, rats, cats,dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, bovines, andnon-human primates (e.g., chimpanzees, macaques, and apes).

“Inhibitors,” “activators,” and “modulators” of expression or ofactivity are used to refer to inhibitory, activating, or modulatingmolecules, respectively, identified using in vitro and in vivo assaysfor expression or activity of a described target protein (or encodingpolynucleotide), e.g., ligands, agonists, antagonists, and theirhomologs and mimetics. The term “modulator” includes inhibitors andactivators. Inhibitors are agents that, e.g., inhibit expression or bindto, partially or totally block stimulation or protease inhibitoractivity, reduce, decrease, prevent, delay activation, inactivate,desensitize, or down regulate the activity of the described targetprotein, e.g., antagonists. Activators are agents that, e.g., induce oractivate the expression of a described target protein or bind to,stimulate, increase, open, activate, facilitate, enhance activation orprotease inhibitor activity, sensitize or up regulate the activity ofdescribed target protein (or encoding polynucleotide), e.g., agonists.Modulators include naturally occurring and synthetic ligands,antagonists and agonists (e.g., small chemical molecules, antibodies andthe like that function as either agonists or antagonists). Such assaysfor inhibitors and activators include, e.g., applying putative modulatorcompounds to cells expressing the described target protein and thendetermining the functional effects on the described target proteinactivity, as described above. Samples or assays comprising describedtarget protein that are treated with a potential activator, inhibitor,or modulator are compared to control samples without the inhibitor,activator, or modulator to examine the extent of effect. Control samples(untreated with modulators) are assigned a relative activity value of100%. Inhibition of a described target protein is achieved when theactivity value relative to the control is about 80%, optionally 50% or25, 10%, 5% or 1%. Activation of the described target protein isachieved when the activity value relative to the control is 110%,optionally 150%, optionally 200, 300%, 400%, 500%, or 1000-3000% or morehigher.

The following statements are intended to describe and summarize variousembodiments of the invention according to the foregoing description inthe specification.

Statements:

-   -   1. A composition comprising a TGFβ family member, a WNT        activator, or a combination thereof.    -   2. The composition of statement 1, further comprising        2-phospho-L-ascorbic acid or vitamin C.    -   3. The composition of statement 1 or 2, wherein the WNT        activator is a lithium salt, CHIR99021, 1-azakenpaullone,        AR-A014418, indirubin-3′-monoxime, 5-Iodo-indirubin-3′-monoxime,        kenpaullone, SB-415286, SB-216763,        2-anilino-5-phenyl-1,3,4-oxadiazole),        (Z)-5-(2,3-Memylenedioxyphenyl)imidazolidine-2,4-dione, TWS119,        CHIR98014, SB415286, Tideglusib, LY2090314, or a combination        thereof    -   4. The composition of any of statements 1-3, wherein the TGFβ        family member is a factor of an Activin/Inhibin subfamily, a        decapentaplegic-Vg-related (DVR) related subfamily (that        includes bone morphogenetic proteins and growth differentiation        factors), and/or a TGF-β subfamily.    -   5. The composition of any of statements 1-4, wherein the TGFβ        family member is Activin A.    -   6. The composition of any of statements 1-5, further comprising        a G9a histone methyltransferase inhibitor selected from the        group consisting of Bix-01294, chaetocin, 3-deazaneplanocin        hydrochloride, UNC 0224, UNC 0638, UNC 0646, and any combination        thereof    -   7. The composition of any of statements 1-6, further comprising        a histone deacetylase inhibitor selected from the group        consisting of sodium butyrate, phenyl butyrate, butyrate,        Suberoylanilide Hydroxamic Acid (SAHA), BML-210, Depudecin, HC        Toxin, Scriptaid, Phenylbutyrate, Valproic Acid, Suramin,        Trichostatin A, APHA Compound 8, Apicidin, Trapoxin B,        Chlamydocin, Depsipeptide, CI-994, MS-27-275, MGCD0103,        NVP-LAQ-824, CBHA, JNJ16241199, Tubacin, A-161906, Proxamide,        Oxamflatin, 3C1-UCHA, AOE, CHAP31, or any combination thereof.    -   8. The composition of any of statements 1-7, further comprising        a DNA methyltransferase inhibitor selected from the group        consisting of RG108, 5-azacitidine, 5-aza-T-deoxycytidine,        decitabine, doxorubicin, EGCG ((−)-epigallocatechin-3-gallate),        zebularine, and any combination thereof    -   9. The composition of any of statements 1-8, further comprising        an adenosine receptor agonist selected from the group consisting        of 5′-N-ethylcarbox-amido-adenosine (NECA),        8-butylamino-adenosine,        2-[p-(2-carboxyethyl)phenethyl-amino]-5′-N-ethylcarboxamidoadenosine        (CGS-2 1680), HENECA,        4-(3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl)-cyclohexanecarboxylic        acid methyl ester), N⁶-cyclopentyladenosine (CPA),        2-chloro-N⁶-cyclopentyl-adenosine (CCPA),        (2S)—N6-[2-endo-Norbornyl]adenosine ((S)-ENBA),        N-(2-aminoethyl)-2-[4-[[2-[4-[[9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]purin-6-yl]amino]phenyl]acetyl]amino]phenyl]acetamide        hydrate (ADAC), AMP579, NNC-21-0136, GR79236, CVT-510        (Tecadenoson), SDZ WAG 994, Selodenoson, and any combination        thereof.    -   10. The composition of any of statements 1-9, wherein the        composition is a cell culture medium or comprises components for        a cell culture medium.    -   11. The composition of any of statements 1-10, wherein the TGFβ        family member, the WNT activator, the 2-phospho-L-ascorbic acid,        the vitamin C, G9a histone methyltransferase inhibitor, histone        deacetylase inhibitor, DNA methyltransferase inhibitor,        adenosine receptor agonist, or a combination thereof, is in an        amount sufficient to generate definitive endodermal cells from        mammalian cells that transiently express pluripotency factors.    -   12. A mixture comprising the composition of any of statements        1-11, and a selected starting mammalian cell population.    -   13. The mixture of statement 12, wherein the mammalian cell        population transiently expresses at least one pluripotency        factor while being incubated in the composition.    -   14. The mixture of statement 12 or 13, wherein the pluripotency        factor comprises OCT4, KLF4, SOX2, or a combination thereof.    -   15. The mixture of statement 14, wherein the pluripotency        further comprises c-MYC, p53 shRNA, or a combination thereof.    -   16. The mixture of any of statements 12-15, wherein the starting        mammalian cell population is non-pluripotent.    -   17. The mixture of any of statements 12-16, wherein the starting        mammalian cell population comprises non-pluripotent fibroblasts,        epidermal cells, lymphocytes, hepatocytes, myoblasts, neurons,        osteoblasts, osteoclasts, and T-cells.    -   18. The mixture of any of statements 12-17, wherein the starting        mammalian cell population comprises partially and/or fully        differentiated cells of epithelial cell lineage, hematopoietic        lineage, endothelial cell lineage, muscle cell lineage, neural        cell lineage, or any combinations thereof.    -   19. The mixture of any of statements 12-18, wherein the starting        mammalian cell population is from a non-pluripotent mammalian        cell population previously contacted with one or more        pluripotency factors or pluripotency inducing agents, or wherein        the non-pluripotent mammalian cell population is induced to        express one or more pluripotency factors.    -   20. The mixture of any of statements 12-19, wherein the starting        mammalian cell population comprises pluripotent stem cells.    -   21. The mixture of any of statements 12-20, wherein the starting        mammalian cell population does not express detectable amounts of        NANOG.    -   22. The mixture of any of statements 12-21, wherein the starting        mammalian cell population is an isolated mammalian cell        population.    -   23. The mixture of any of statements 12-22, wherein the starting        mammalian cell population is an isolated allogeneic mammalian        cell population.    -   24. The mixture of any of statements 12-23, wherein the starting        mammalian cell population is an autologous mammalian cell        population isolated from a patient.    -   25. The mixture of any of statements 12-24, wherein the starting        mammalian cell population is contacted with the composition for        about 4 days to about 28 days.    -   26. The mixture of any of statements 12-25, wherein a portion of        the starting mammalian cell population is converted into        definitive endodermal cells after about 7 days, or after about        10 days, or after about 12 days, or after about 14 days, or        after about 16 days, or after about 18 days, or after about 20        days of contact with the composition.    -   27. The mixture of any of statements 12-26, wherein a portion of        the starting mammalian cell population express Sox17, Foxa2,        Cerberus 1 (Cer), C—X—C chemokine receptor type 4 (Cxcr4), or a        combination thereof, after about 7 days, or after about 10 days,        or after about 12 days, or after about 14 days, or after about        16 days, or after about 18 days, or after about 20 days of        contact with the composition.    -   28. The mixture of any of statements 12-27, wherein the starting        mammalian cell population and cells thereof that convert into        definitive endodermal cells do not express detectable levels of        NANOG.    -   29. A composition comprising a TGFβ receptor inhibitor, a        hedgehog pathway inhibitor, and a retinoic acid receptor        agonist.    -   30. The composition of statement 29, further comprising        epidermal growth factor (EGF), basic fibroblast growth factor        (bFGF), fibroblast growth factor 7 (FGF7), fibroblast growth        factor 10 (FGF10), or a combination thereof    -   31. The composition of statement 29 or 30, further comprising a        WNT activator, 2-phospho-L-ascorbic acid, vitamin C, a Notch        signaling inhibitor, a BMP4 signaling inhibitor, or a        combination thereof    -   32. The composition of any of statements 29-31, wherein the TGFβ        receptor inhibitor, the hedgehog pathway inhibitor, the retinoic        acid receptor agonist, the WNT activator, the        2-phospho-L-ascorbic acid, the vitamin C, the Notch signaling        inhibitor, the BMP4 signaling inhibitor, or any combination        thereof, is in an amount sufficient to generate pancreatic        progenitor cells from mammalian cells that express Sox17, Foxa2,        Cerberus 1 (Cer), C—X—C chemokine receptor type 4 (Cxcr4), or a        combination thereof.    -   33. The composition of any of statements 29-32, wherein the TGFβ        receptor inhibitor is selected from the group consisting of        A83-01, SB431542, SB 431542, SJN 2511, D 4476, LY 364947,        SB505124, SB 525334, SD 208, LDN-193189, and any combination        thereof    -   34. The composition of any of statements 29-33, wherein the        hedgehog pathway inhibitor is selected from the group consisting        of LDE 225, cyclopamine, MK-4101, GDC-0449, XL-139 (BMS-833923),        PF-04449913, robotnikinin, Cur-61414 (G-024856), and any        combination thereof    -   35. The composition of any of statements 29-34, wherein the        retinoic acid receptor agonist is selected from the group        consisting of retinoic acid, 9-cis-retinoic acid, honokiol,        magnolol Am80, AM580, TTNPB, AC55649, and any combination        thereof.    -   36. The composition of any of statements 29-35, wherein the WNT        activator is a lithium salt, CHIR99021, 1-azakenpaullone,        AR-A014418, indirubin-3′-monoxime, 5-Iodo-indirubin-3′-monoxime,        kenpaullone, SB-415286, SB-216763,        2-anilino-5-phenyl-1,3,4-oxadiazole),        (Z)-5-(2,3-Memylenedioxyphenyl)imidazolidine-2,4-dione, TWS119,        CHIR98014, SB415286, Tideglusib, LY2090314, or a combination        thereof.    -   37. The composition of any of statements 29-36, wherein the        Notch signaling inhibitor is Compound E, R04929097, DAPT        (GSI-IX), Gamma-Secretase Inhibitor I (Z-Leu-Leu-Nle-CHO where        Nle is Norleucine), Gamma-Secretase Inhibitor II, or any        combination thereof.    -   38. The composition of any of statements 29-37, wherein the BMP4        signaling inhibitor is noggin, chordin, CeM, DAN, Gremlin,        K02288, LDN-193189, or any combination thereof.    -   39. The composition of any of statements 29-38, wherein the        composition is a cell culture medium or comprises components for        a cell culture medium.    -   40. A mixture comprising the composition of any of statements        29-39, and a selected mammalian cell population.    -   41. The mixture of statement 40, wherein the selected mammalian        cell population comprises cells that express Sox17, Foxa2,        Cerberus 1 (Cer), C—X—C chemokine receptor type 4 (Cxcr4), or a        combination thereof.    -   42. The mixture of statement 40 or 41, wherein the selected        mammalian cell population comprises at least 5%, or at least        10%, or at least 12%, or at least 15%, or at least 25%, or at        least 50%, or at least 60%, or at least 70%, or at least 80%, or        at least 90%, or at least 95% cells that express Sox17, Foxa2,        Cerberus 1 (Cer), C—X—C chemokine receptor type 4 (Cxcr4), or a        combination thereof.    -   43. The mixture of any of statements 40-43, wherein the selected        mammalian cell population is formed from a nonpluripotent cell        population or a stem cell population redirected to express        Sox17, Foxa2, Cerberus 1 (Cer), C—X—C chemokine receptor type 4        (Cxcr4), or a combination thereof    -   44. The mixture of any of statements 40-43, wherein the selected        mammalian cell population is formed from cells contacted with a        first composition comprising a TGFβ family member, a WNT        activator, or a combination thereof    -   45. The mixture of statement 44, wherein the first composition        further comprises 2-phospho-L-ascorbic acid or vitamin C.    -   46. The mixture of statement 44 or 45, wherein the first        composition has the TGFβ family member, the WNT activator, the        2-phospho-L-ascorbic acid, the vitamin C, or a combination        thereof, in an amount sufficient to generate definitive        endodermal cells.    -   47. The mixture of any of statements 40-46, wherein when        initially mixed with the composition, cells in the selected        mammalian cell population do not express Rex1, Nanog, or a        combination thereof    -   48. The mixture of any of statements 40-47, wherein a portion of        the selected mammalian cell population is converted into        pancreatic progenitor cells after 4 hours, or after about 8        hours, or after about 12 hours, or after about 1 day, or after        about 2 days, or after about 3 days, or after about 4 days, or        after about 5 days, or after about 6 days of contact with the        composition.    -   49. The mixture of any of statements 40-48, wherein after mixing        the selected mammalian cell population with the composition for        at least one day, cells in the selected mammalian cell        population express Pdx1, Nkx6.1, or a combination thereof.    -   50. The mixture of any of statements 40-49, wherein after mixing        the selected mammalian cell population with the composition for        2 or more days, cells in the selected mammalian cell population        express Pdx1, Nkx6.1, Pax6, Hnf6, or a combination thereof    -   51. The mixture of any of statements 40-50, wherein a portion of        the selected mammalian cell population is converted into        pancreatic progenitor cells after 4 hours, or after about 8        hours, or after about 12 hours, or after about 1 day, or after        about 2 days, or after about 3 days, or after about 4 days, or        after about 5 days, or after about 6 days of contact with the        composition.    -   52. The mixture of any of statements 40-51, wherein after mixing        the selected mammalian cell population with the composition for        2 or more days, at least 5%, or at least about 10%, or at least        about 12%, or at least about 16%, or at least about 17%, or at        least about 20%, or at least about 25% of cells in the selected        mammalian cell population express Pdx1, Nkx6.1, Pax6, Hnf6, or a        combination thereof    -   53. A method comprising, contacting a selected starting        mammalian cell population with a composition comprising a TGFβ        family member, a WNT activator, or a combination thereof    -   54. The method of statement 53, wherein the composition further        comprises 2-phospho-L-ascorbic acid or vitamin C.    -   55. The method of statement 53 or 54, wherein the WNT activator        is a lithium salt, CHIR99021, 1-azakenpaullone, AR-A014418,        indirubin-3′-monoxime, 5-Iodo-indirubin-3′-monoxime,        kenpaullone, SB-415286, SB-216763,        2-anilino-5-phenyl-1,3,4-oxadiazole),        (Z)-5-(2,3-Memylenedioxyphenyl)imidazolidine-2,4-dione, TWS119,        CHIR98014, SB415286, Tideglusib, LY2090314, or a combination        thereof    -   56. The method of any of statements 53-55, wherein the TGFβ        family member is a factor of an Activin/Inhibin subfamily, a        decapentaplegic-Vg-related (DVR) related subfamily (that        includes bone morphogenetic proteins and growth differentiation        factors), and/or a TGF-β subfamily.    -   57. The method of any of statements 53-56, wherein the TGFβ        family member is Activin A.    -   58. The method of any of statements 53-57, wherein the        composition further comprises a G9a histone methyltransferase        inhibitor selected from the group consisting of Bix-01294,        chaetocin, 3-deazaneplanocin hydrochloride, UNC 0224, UNC 0638,        UNC 0646, and any combination thereof    -   59. The method of any of statements 53-58, wherein the        composition further comprises a histone deacetylase inhibitor        selected from the group consisting of sodium butyrate, phenyl        butyrate, butyrate, Suberoylanilide Hydroxamic Acid (SAHA),        BML-210, Depudecin, HC Toxin, Scriptaid, Phenylbutyrate,        Valproic Acid, Suramin, Trichostatin A, APHA Compound 8,        Apicidin, Trapoxin B, Chlamydocin, Depsipeptide, CI-994,        MS-27-275, MGCD0103, NVP-LAQ-824, CBHA, JNJ16241199, Tubacin,        A-161906, Proxamide, Oxamflatin, 3Cl-UCHA, AOE, CHAP31, or any        combination thereof    -   60. The method of any of statements 53-59, wherein the        composition further comprises a DNA methyltransferase inhibitor        selected from the group consisting of RG108, 5-azacitidine,        5-aza-T-deoxycytidine, decitabine, doxorubicin, EGCG        ((−)-epigallocatechin-3-gallate, zebularine, and any combination        thereof    -   61. The method of any of statements 53-60, wherein the        composition further comprises an adenosine receptor agonist        selected from the group consisting of        5′-N-ethylcarbox-amido-adenosine (NECA), 8-butylamino-adenosine,        2-[p-(2-carboxyethyl)phenethyl-amino]-5′-N-ethylcarboxamidoadenosine        (CGS-2 1680), HENECA,        4-(3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl)-cyclohexanecarboxylic        acid methyl ester), N⁶-cyclopentyladenosine (CPA),        2-chloro-N⁶-cyclopentyl-adenosine (CCPA),        (2S)—N6-[2-endo-Norbornyl]adenosine ((S)-ENBA),        N-(2-aminoethyl)-2-[4-[[2-[4-[[9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]purin-6-yl]amino]phenyl]acetyl]amino]phenyl]acetamide        hydrate (ADAC), AMP579, NNC-21-0136, GR79236, CVT-510        (Tecadenoson), SDZ WAG 994, Selodenoson, and any combination        thereof.    -   62. The method of any of statements 53-61, wherein the TGFβ        family member, the WNT activator, the 2-phospho-L-ascorbic acid,        the vitamin C, G9a histone methyltransferase inhibitor, histone        deacetylase inhibitor, DNA methyltransferase inhibitor,        adenosine receptor agonist, or a combination thereof, is in an        amount sufficient to generate definitive endodermal cells from a        mammalian cell population that transiently expresses        pluripotency factors.    -   63. The method of any of statements 53-62, wherein the TGFβ        family member, the WNT activator, the 2-phospho-L-ascorbic acid,        the vitamin C, G9a histone methyltransferase inhibitor, histone        deacetylase inhibitor, DNA methyltransferase inhibitor,        adenosine receptor agonist, or a combination thereof, is in an        amount sufficient to generate mammalian cells that express        Sox17, Foxa2, Cerberus 1 (Cer), C—X—C chemokine receptor type 4        (Cxcr4), or a combination thereof, from a mammalian cell        population that transiently expresses pluripotency factors.    -   64. The method of any of statements 53-63, wherein the starting        mammalian cell population transiently expresses at least one        pluripotency factor while being incubated in the composition.    -   65. The method of statement 64, wherein the pluripotency factor        comprises OCT4, KLF4, SOX2, or a combination thereof.    -   66. The method of statement 64 or 65, wherein the pluripotency        further comprises c-MYC, p53 shRNA, or a combination thereof.    -   67. The method of any of statements 53-66, wherein the starting        mammalian cell population is non-pluripotent.    -   68. The method of any of statements 53-67, wherein the starting        mammalian cell population comprises non-pluripotent fibroblasts,        epidermal cells, lymphocytes, hepatocytes, myoblasts, neurons,        osteoblasts, osteoclasts, T-cells, or any combination thereof    -   69. The method of any of statements 53-68, wherein the starting        mammalian cell population comprises partially and/or fully        differentiated cells of epithelial cell lineage, hematopoietic        lineage, endothelial cell lineage, muscle cell lineage, neural        cell lineage, or any combination thereof    -   70. The method of any of statements 53-69, wherein the starting        mammalian cell population is from a non-pluripotent mammalian        cell population previously contacted with one or more        pluripotency factors or pluripotency inducing agents, or wherein        the non-pluripotent mammalian cell population is induced to        express one or more pluripotency factors.    -   71. The method of any of statements 53-68, wherein the starting        mammalian cell population comprises pluripotent stem cells.    -   72. The method of any of statements 53-70, wherein the starting        mammalian cell population does not express detectable amounts of        NANOG.    -   73. The method of any of statements 53-72, wherein the starting        mammalian cell population is an isolated mammalian cell        population.    -   74. The method of any of statements 53-73, wherein the starting        mammalian cell population is an isolated allogeneic mammalian        cell population.    -   75. The method of any of statements 53-74, wherein the starting        mammalian cell population is an autologous mammalian cell        population isolated from a patient.    -   76. The method of any of statements 53-75, wherein the starting        mammalian cell population is contacted with the composition for        about 4 days to about 28 days.    -   77. The method of any of statements 53-76, wherein a portion of        the starting mammalian cell population is converted into        definitive endodermal cells after about 7 days, or after about        10 days, or after about 12 days, or after about 14 days, or        after about 16 days, or after about 18 days, or after about 20        days of contact with the composition.    -   78. The method of any of statements 53-77, wherein a portion of        the starting mammalian cell population express Sox17, Foxa2,        Cerberus 1 (Cer), C—X—C chemokine receptor type 4 (Cxcr4), or a        combination thereof, after about 7 days, or after about 10 days,        or after about 12 days, or after about 14 days, or after about        16 days, or after about 18 days, or after about 20 days of        contact with the composition.    -   79. The method of any of statements 53-78, wherein the starting        mammalian cell population and cells converting therefrom into        definitive endodermal cells do not express detectable levels of        NANOG.    -   80. The method of any of statements 53-79, further comprising        administering cells generated by the method to a mammal    -   81. The method of any of statements 53-80, further comprising        administering to a mammal at least some definitive endodermal        cells generated by the method.    -   82. The method of any of statements 53-81, further comprising        administering cells generated by the method to a mammal, wherein        the cells express Sox17, Foxa2, Cerberus 1 (Cer), C—X—C        chemokine receptor type 4 (Cxcr4), or a combination thereof.    -   83. A method comprising contacting a cell population comprising        Sox17⁺, Foxa2⁺ cells with one or more growth factors, WNT        activators, TGFβ receptor inhibitors, or a combination thereof        for a time sufficient to expand the cell number by at least        ten-fold and thereby generate an expanded population of        posterior foregut-like progenitor cells.    -   84. The method of statement 83, wherein the expanded population        of posterior foregut-like progenitor cells maintains an        epithelial colony morphology.    -   85. The method of statement 83 or 84, wherein the expanded        population of posterior foregut-like progenitor cells express        SOX17, FOXA2, HNF4a, HNF6, SOX9, or any combination thereof    -   86. The method of any of statements 83-85, wherein the one or        more growth factors is a combination of EGF and bFGF.    -   87. The method of any of statements 83-86, wherein the WNT        activator is a lithium salt, CHIR99021, 1-azakenpaullone,        AR-A014418, indirubin-3′-monoxime, 5-Iodo-indirubin-3′-monoxime,        kenpaullone, SB-415286, SB-216763,        2-anilino-5-phenyl-1,3,4-oxadiazole),        (Z)-5-(2,3-Memylenedioxyphenyl)imidazolidine-2,4-dione, TWS119,        CHIR98014, SB415286, Tideglusib, LY2090314, or a combination        thereof    -   88. The method of any of statements 83-87, wherein the WNT        activator is CHIR99021.    -   89. The method of any of statements 83-88, wherein the TGFβ        receptor inhibitor is selected from the group consisting of        A83-01, SB431542, SB 431542, SJN 2511, D 4476, LY 364947,        SB505124, SB 525334, SD 208, LDN-193189, and any combination        thereof    -   90. The method of any of statements 83-89, wherein the TGFβ        receptor inhibitor is A83-01.    -   91. The method of any of statements 83-90, wherein the cell        number is expanded by at least 100-fold, or at least 1000-fold,        or at least 10,000-fold, or at least 100,000-fold, or by at        least 1 million-fold, or by at least 10 million-fold, or by at        least 100 million-fold.    -   92. The method of any of statements 83-91, further comprising        converting the posterior foregut-like progenitor cells into        pancreatic progenitor cells.    -   93. The method of any of statements 83-92, further comprising        contacting the posterior foregut-like progenitor cells with a        composition comprising at least one FGF7, FGF10, TGFβ receptor        inhibitor, Notch signaling inhibitor, retinoic acid receptor        agonist, hedgehog inhibitor, BMP4 signaling inhibitor, or a        combination thereof for at least one day to generate        differentiating cells.    -   94. The method of statement 93, further comprising contacting        the differentiating cells with EGF, a glucagon-like peptide-1        agonist, a TGFβ receptor inhibitor, a BMP4 signaling inhibitor,        an activator of protein kinase C, a Notch signaling inhibitor, a        polyADP-ribose synthetase inhibitor, or a combination thereof,        for at least 2 days to generate a population of pancreatic        progenitor cells.    -   95. The method of any of statements 83-94, wherein the        pancreatic progenitor cells express FOXA2, HNF6, SOX9, PDX1, or        any combination thereof.    -   96. The method of any of statements 83-95, further comprising        administering the posterior foregut-like progenitor cells, the        differentiating cells, the pancreatic progenitor cells, or a        combination thereof, to a mammalian subject.    -   97. The method of any of statements 83-96, wherein the mammalian        subject has diabetes.    -   98. A method comprising contacting pancreatic progenitor cells        with a composition comprising vitamin C (or phospho-L-ascorbic        acid) and nicotinamide to generate a population of pancreatic        beta cells.    -   99. The method of statement 98, wherein the pancreatic        progenitor cells express FOXA2, HNF6, SOX9, PDX1, or any        combination thereof.    -   100. The method of statement 98 or 99, wherein the pancreatic        beta cells express insulin, glucagon, PDX1, NKX6.1, NEUROD1,        NKX2.2, or any combination thereof.    -   101. The method of any of statements 98-100, wherein the        composition further comprises a TGFβ receptor inhibitor, a p38        mitogen-activated protein (MAP) kinase inhibitor, a Notch        signaling inhibitor, a glucagon-like peptide-1 agonist, a        basement membrane protein, an adenylyl cyclase activator, a        glucocorticoid receptor agonist, Ca′ channel agonist, or any        combination thereof    -   102. A method comprising: contacting pancreatic endodermal        progenitor cells with a composition comprising at least five of        the following: heparin, zinc salts, a TGF-β inhibitor, a BMP4        signaling inhibitor, a T3 thyroid hormone, cysteine, an        antioxidant, an Axl kinase inhibitor, a Notch Signaling        Inhibitor, a Ca²⁺ channel agonist, vitamin C, or any combination        thereof to generate a population of partially or fully        differentiated pancreatic beta-like cells.    -   103. The method of statement 102, wherein at least six, or at        least seven, or at least eight of the following: heparin, zinc        salts, a TGF-β inhibitor, a BMP4 signaling inhibitor, a T3        thyroid hormone, cysteine, an antioxidant, an Axl kinase        inhibitor, a Notch Signaling Inhibitor, a Ca²⁺ channel agonist,        vitamin C, or any combination thereof are contacted with the        pancreatic endodermal progenitor cells.    -   104. A method comprising: (a) contacting pancreatic endodermal        progenitor cells with a first composition that includes heparin,        zinc salts, a TGF-β inhibitor, a BMP4 signaling inhibitor, a T3        thyroid hormone, a Notch Signaling Inhibitor, a Ca²⁺ channel        agonist, vitamin C, and combinations thereof to generate a first        population of partially differentiated pancreatic beta-like        cells; (b) contacting the first population of cells with a        second composition that includes heparin zinc salts, a TGF-β        inhibitor, a BMP4 signaling inhibitor, a T3 thyroid hormone,        cysteine, an anti-oxidant, an Axl kinase inhibitor, a Ca²⁺        channel agonist, vitamin C, or any combination thereof to        generate a second population of pancreatic beta-like cells.    -   105. The method of any of statements 102-104 further comprising        step (c) wherein the second population of cells is cultured to        generate 3D aggregates using low-attachment plates to generate        3D pancreatic beta cells.    -   106. The method of any of statements 102-105, wherein the        pancreatic beta cells or the 3D pancreatic beta cells are        administered to a mammal    -   107. A method comprising:        -   a. contacting pancreatic endodermal progenitor cells with a            composition comprising vitamin C, BayK-8644, heparin, zinc            sulfate, Alk5 inhibitor II, LDN-193189, T3 thyroid hormone,            and Compound E for three to fifteen days to generate a first            population of cells;        -   b. contacting the first population of cells with a second            composition comprising vitamin C, BayK-8644, heparin, zinc            sulfate, Alk5 inhibitor II, T3 thyroid hormone, N-cysteine,            Trolox, and R428 for three to fifteen days to generate a            second population of cells; and        -   c. culturing the second population of cells as 3D aggregates            in low-attachment plates for three to fifteen days to            thereby generate a third cell population comprising            functional pancreatic beta-like cells.    -   108. The method of statement 107, further comprising        administering the functional pancreatic beta-like cells to a        mammal in need thereof    -   109. The method of statement 106 or 108, wherein the mammal has        type I diabetes, type II diabetes, or type 1.5 diabetes.    -   110. A method of producing a pancreatic beta cell from a stem        cell comprising:        -   (a) Exposing a stem cell to 10-300 ng/ml Epidermal Growth            Factor or 10-300 ng/ml Keratinocyte Growth Factor for 12-72            hours in DMEM comprising 0.1-5 mM glutamine (0.05-2.5X            GlutaMAX™), 0.1-5X (e.g., 1X) Invitrogen non-essential amino            acids, and 0.1-5X (e.g., 1X) B27 supplement under conditions            suitable for cell culture growth, thereby maintaining a            progenitor cell;        -   (b) incubating the progenitor cell in DMEM comprising 0.1-5X            (e.g., 1X) Invitrogen non-essential amino acids, 0.1-5 mM            glutamine (0.05-2.5X GlutaMAX™), 2-20 μg/ml heparin, 0.2-5            mM cysteine, 2-20 μM zinc, 2-20 μM ALK inhibitor, 0.2-2 μM            BMP inhibitor LDN-193189, 0.2-5 μM T3 thyroid hormone and            0.2-5 μM gamma secretase inhibitor XX to yield a cell in            culture; and        -   (c) adding to the cell in culture 10-2000 μM vitamin C and            0.2-5 μM BayK-8644, thereby producing a functional            pancreatic beta cell.    -   111. The method of statement 110 wherein the stem cell is        -   (a) exposed to 50 ng/ml Epidermal Growth Factor or 50 ng/ml            of Keratinocyte Growth Factor for 12-72 hours in DMEM            comprising 2 mM glutamine (1X GlutaMAX™), 0.1-5X (e.g., 1X)            Invitrogen non-essential amino acids, and 0.1-5X (e.g., 1X)            B27 supplement;        -   (b) incubated in DMEM comprising 0.1-5X (e.g., 1X)            Invitrogen non-essential amino acids, 2 mM glutamine (1X            GlutaMAX™), 10 μg/ml heparin, 1 mM cysteine, 10 μM zinc, 10            μM ALK inhibitor, 0.5 μM BMP inhibitor LDN-193189, 1 μM T3            thyroid hormone and 1 μM gamma secretase inhibitor XX to            yield a cell in culture; and        -   (c) adding to the cell in culture 500 μM vitamin C and 2 μM            BayK-8644, thereby producing a functional pancreatic beta            cell.    -   112. The method of statement 111 wherein the stem cell is        exposed to Epidermal Growth Factor or Keratinocyte Growth Factor        for 24-48 hours.    -   113. The method of any one of statements 110-112 wherein the        stem cell is an embryonic stem cell.    -   114. The method of any one of statements 110-113 wherein the        stem cell is a human stem cell.    -   115. The method of any one of statements 110-114 wherein the        stem cell is exposed to epidermal growth factor.    -   116. The method of any one of statements 110-115 wherein the        stem cell is exposed to keratinocyte growth factor.    -   117. The method of any one of statements 110-114 wherein the        stem cell is exposed to epidermal growth factor and keratinocyte        growth factor.    -   118. The method of any one of statements 110-117 wherein the        functional pancreatic beta cell exhibits a 1-7-fold increase in        insulin secretion upon stimulation with glucose.    -   119. The method of any one of statements 110-118 wherein the        pancreatic beta cell is functional immediately upon        transplantation.    -   120. The method of any one of statements 110-119 wherein the        pancreatic beta cell is functional within one week of        transplantation.    -   121. The method of any one of statements 110-120 wherein the        pancreatic beta cell remains functional for at least four weeks.    -   122. The method of any one of statements 110-121 further        comprising exposure of the stem cell to an oxygen level no        greater than 10% O₂.    -   123. The method of statement 122 wherein the oxygen level is no        greater than 5% O₂.    -   124. The method of statement 122 wherein the oxygen level is no        greater than 4% O₂.    -   125. The method of any one of statements 110-124 wherein NGN3        and PDX1/NKX6.1 are expressed during the incubating step and the        adding step.    -   126. The method of any one of statements 110-125 wherein at        least 75% of the stem cells differentiate into functional        pancreatic beta cells.    -   127. The method of any one of statements 110-126 wherein the        progenitor cell is maintained for up to 32 days in culture        containing Epidermal Growth Factor or Keratinocyte Growth        Factor.    -   128. A functional pancreatic beta cell produced according to the        method of any one of statements 110-127.    -   129. The functional pancreatic beta cell of statement 128,        wherein the cell is a human cell.    -   130. The functional pancreatic beta cell of statement 128 or        statement 129, wherein the cell expresses at least three        pancreatic cell markers selected from the group of human        c-peptide (C-PEP), Chromagranin A (CHGA), transcription factor        NKX6.1, transcription factor PDX1, transcription factor PAX6,        transcription factor NKX2.2, transcription factor NEUROD1 and        transcription factor ISL1 without inducing expression of        Glucagon (GCG) or Somatostatin (SST).    -   131. A method for treating diabetes comprising administering an        effective amount of the cell according to any of statements        110-130 to a diabetic subject.    -   132. The method of statement 131 wherein the subject is human.    -   133. A method of producing a pancreatic beta cell from a stem        cell comprising:        -   (a) exposing a stem cell to Epidermal Growth Factor or            Keratinocyte Growth Factor for 12-72 hours under conditions            suitable for cell culture growth, thereby maintaining a            progenitor cell;        -   (b) incubating the progenitor cell in a culture medium            comprising heparin, cysteine, zinc, ALK inhibitor, BMP            inhibitor LDN-193189, T3 thyroid hormone and gamma secretase            inhibitor XX to yield a cell in culture; and        -   (c) adding to the cell in culture vitamin C and BayK-8644,            thereby producing a functional pancreatic beta cell.    -   134. The method of statement 133, wherein the stem cell of        step (a) is exposed to 10-300 ng/ml Epidermal Growth Factor or        10-300 ng/ml of Keratinocyte Growth Factor; the culture medium        of step (b) comprises 2-20 μg/ml heparin, 0.2-5 mM cysteine,        2-20 μM zinc, 2-20 μM ALK inhibitor, 0.2-2 μM BMP inhibitor        LDN-193189, 0.2-5 μM T3 thyroid hormone and 0.2-5 μM gamma        secretase inhibitor XX; and step (c) comprises adding to the        cell in culture 10-2000 μM vitamin C and 0.2-5 μM BayK-8644.    -   135. The method of statement 134, wherein the stem cell of        step (a) is exposed to 50 ng/ml Epidermal Growth Factor or 50        ng/ml of Keratinocyte Growth Factor; the culture medium of        step (b) comprises 10 μg/ml heparin, 1 mM cysteine, 10 μM zinc,        10 μM ALK inhibitor, 0.5 μM BMP inhibitor LDN-193189, 1 μM T3        thyroid hormone and 1 μM gamma secretase inhibitor XX; and        step (c) comprises adding to the cell in culture 500 μM vitamin        C and 2 μM BayK-8644.    -   136. The method of any one of statements 133-135, wherein said        exposing of step (a) is in DMEM comprising 0.1-5 mM glutamine or        0.05-2.5X GlutaMAX™, 0.1-5X non-essential amino acids, and        0.1-5X B27 supplement.    -   137. The method of any one of statements 133-136, wherein the        culture medium of step (b) comprises DMEM comprising 0.1-5 mM        glutamine or 0.05-2.5X GlutaMAX™, and 0.1-5X non-essential amino        acids.    -   138. The method of any one of statements 133-137, wherein said        exposing of step (a) is in DMEM comprising 2 mM glutamine or 1X        GlutaMAX™.    -   139. The method of any one of statements 133-138, wherein the        culture medium of step (b) comprises DMEM comprising 2 mM        glutamine or 1X GlutaMAX™.    -   140. The method of any one of statements 133-139, wherein the        stem cell is exposed to Epidermal Growth Factor or Keratinocyte        Growth Factor for 24-48 hours.    -   141. The method of any one of statements 133-140, wherein the        stem cell is an embryonic stem cell.    -   142. The method of any one of statements 133-141, wherein the        stem cell is a human stem cell.    -   143. The method of any one of statements 133-142, wherein the        stem cell is exposed to epidermal growth factor.    -   144. The method of any one of statements 133-143, wherein the        stem cell is exposed to keratinocyte growth factor.    -   145. The method of any one of statements 133-142, wherein the        stem cell is exposed to epidermal growth factor and keratinocyte        growth factor.    -   146. The method of any one of statements 133-145, wherein the        functional pancreatic beta cell exhibits a 1-7-fold increase in        insulin secretion upon stimulation with glucose.    -   147. The method of any one of statements 133-145, wherein the        functional pancreatic beta cell exhibits a 2-fold or more        increase in insulin secretion upon stimulation with glucose.    -   148. The method of any one of statements 133-147, wherein the        pancreatic beta cell is functional immediately upon        transplantation.    -   149. The method of any one of statements 133-148, wherein the        pancreatic beta cell is functional within one week of        transplantation.    -   150. The method of any one of statements 133-149, wherein the        pancreatic beta cell remains functional for at least four weeks.    -   151. The method of any one of statements 133-150, further        comprising exposure of the stem cell to an oxygen level no        greater than 10% O₂.    -   152. The method of statement 151, wherein the oxygen level is no        greater than 5% O₂.    -   153. The method of statement 152, wherein the oxygen level is no        greater than 4% O₂.    -   154. The method of any one of statements 133-153, wherein NGN3        and PDX1/NKX6.1 are expressed during the incubating step and the        adding step.    -   155. The method of any one of statements 133-154, wherein at        least 75% of the stem cells differentiate into functional        pancreatic beta cells.    -   156. The method of any one of statements 133-155, wherein the        progenitor cell is maintained for up to 32 days in culture        containing Epidermal Growth Factor or Keratinocyte Growth        Factor.    -   157. A functional pancreatic beta cell produced according to the        method of any one of statements 133-156.    -   158. The functional pancreatic beta cell of statement 157,        wherein the cell is a human cell.    -   159. The functional pancreatic beta cell of statement 157 or        statement 158, wherein the cell expresses at least three        pancreatic cell markers selected from the group of human        c-peptide (C-PEP), Chromagranin A (CHGA), transcription factor        NKX6.1, transcription factor PDX1, transcription factor PAX6,        transcription factor NKX2.2, transcription factor NEUROD1 and        transcription factor ISL lwithout inducing expression of        Glucagon (GCG) or Somatostatin (SST).    -   160. A method for treating diabetes comprising administering an        effective amount of the cell according to any one of claims        157-159, to a diabetic subject.    -   161. The method of statement 160, wherein the subject is human.    -   162. Any of the above statements, wherein the pancreatic beta        cells, functional pancreatic beta cells, or functional        pancreatic beta-like cells express one or more of insulin,        glucagon, PDX1, NKX6.1, NEUROD1, and NKX2.2.    -   163. Any of the above statements, wherein the pancreatic beta        cells, functional pancreatic beta cells, or functional        pancreatic beta-like cells express at least three pancreatic        cell markers selected from the group of human c-peptide (C-PEP),        Chromagranin A (CHGA), transcription factor NKX6.1,        transcription factor PDX1, transcription factor PAX6,        transcription factor NKX2.2, transcription factor NEUROD1 and        transcription factor ISL1 without inducing expression of        Glucagon (GCG) or Somatostatin (SST).

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Materials and Methods

This Example describes some of the materials and methods employed indevelopment of the invention.

Dox-Inducible Secondary MEF Preparation

NGFP1 Dox-inducible induced pluripotent stem (iPS) cells (Stemgent) wereinjected into C57BL/6 blastocysts and implanted into surrogate mice(CD1, Harlan). Chimeric embryos were isolated at E12.5-13.5. Heads,developing organs and spinal cords were carefully removed from theembryos, and MEFs were prepared and specifically selected as reported(Hanna, Markoulaki et al. 2008; Wernig, Lengner et al. 2008; Hanna, Sahaet al. 2009).

Direct Reprogramming of Fibroblasts into Definitive Endoderm-Like Cells

Dox-inducible secondary mouse embryonic fibroblasts (MEFs) (cells atpassage 3-4 were regularly used) were plated on Matrigel-coated culturedishes at a cell density of 1×10⁴ cells/cm². Cells were cultured in MEFmedium (Dulbecco's modified Eagle's medium with 10% fetal bovine serumand 2 mM Glutamax) for an additional day. The medium was then changed toMed I (Knock-out DMEM with 10% knock-out serum replacer, 5% FBS, 2 mMGlutamax, 0.1 mM non-essential amino acids (NEAA), and 0.055 mMβ-mercaptoethanol) supplemented with 4 μg/ml Dox, 50 ng/ml Activin A and1 mM LiCl for the indicated time. Thereafter, the medium was changed toMed II (75% Iscove's modified Dulbecco's medium, 25% Ham's F12 medium,supplemented with 1×N2 supplements, 0.05% bovine serum albumin, 2 mMGlutamax, and 0.45 mM monothiolglycerol (MTG)) supplemented with 50ng/ml Activin A and 1 mM LiCl for the indicated time. All reagents werepurchased from Invitrogen if not specified, and all cytokines were fromR&D Systems.

Differentiation of Definitive Endoderm-Like Cells (DELCs) to PancreaticProgenitor-Like Cells (PPLCs)

DELCs were further cultured in Med III (DMEM supplemented with 1xB27, 2mM Glutamax), and treated with 2 μM retinoic acid (RA), 1 μM A83-01, 2μM LDE225 and 280 μM 2-phospho-L-ascorbic acid trisodium salt (pVc) for1 day, and then with 1 μM A83-01, 2 μM LDE225 and 280 μM pVc for another3 days. At the end of treatment, lineage specific markers were analyzedby immunostaining and/or real-time PCR.

Immunocytochemistry

Immunocytochemistry analysis was performed as previously described (Efeet al. 2011). Primary antibodies used were Sox17 (R&D), Foxa2(Millipore), Pdx1 (R&D), Hnf6 (Santa Cruz), Pax6 (Covance), Sox9 (SantaCruz), Nkx6.1 (Developmental Studies Hybridoma Bank), Insulin (Dako),Glucagon (Sigma), Somatostatin (Abeam), Amylase (Abeam), and CK19(Abcam). Nuclei were visualized by DAPI (Sigma-Aldrich) staining Imageswere captured using a Nikon Eclipse TE2000-U microscope.

Gene Expression Analysis by Real-Time PCR

Total RNA was extracted using the RNeasy Plus Mini Kit in combinationwith QIAshredder (Qiagen). First-strand reverse transcription wasperformed with 2 μg RNA with the iScript™ cDNA Synthesis Kit (BioRad).Expression of pluripotency and lineage-specific marker genes wasanalyzed by real-time PCR with iQ SYBR Green Supermix (Bio-Rad).

Transplantation Assay

Male, 8-10-week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (JacksonLaboratory, Bar Harbor, Me.) were injected intraperitoneally with 35mg/kg Streptozocin (STZ; Sigma-Aldrich, St. Louis, Mo.) daily for 4days. Blood glucose levels were assayed from tail-vein blood with astandard blood glucose meter (Diabetic Care Services, Eastlake, Ohio).Hyperglycemia developed 5-7 days after the STZ injection. Mice wereconsidered to be diabetic if blood glucose measurements were >300 mg/dlfor 4 consecutive days, after which they were used as transplantrecipients. Under anesthesia, diabetic mice received a renal subcapsulartransplant of about 3×10⁶ pancreatic progenitor-like cells (n=14) or3×10⁶ secondary MEF cells (n=10). Untreated normal mice (n=4) and micetreated with STZ only (n=4) were used as controls. Non-fasting bloodglucose levels were measured weekly after surgery for 8 weeks. Seruminsulin was measured by ELISA (Millipore, Billerica, Mass.) at the endof 8 weeks. All animal work was approved by the institutional IACUCcommittee.

Immunohistochemistry

Left kidneys were removed from euthanized mice, fixed in 4%paraformaldehyde and used for paraffin section and cryosection. Kidneyswere transversally sectioned and stained with primary antibodies. Then,the antigen-primary antibody immune complex was visualized withfluorescent secondary antibodies (Invitrogen). Cell nuclei werecounterstained with DAPI.

Insulin Detection Assay

An insulin-release assay was performed as described (Schroeder et al.2006). Briefly, cells were washed five times with PBS and pre-incubatedin freshly prepared Krebs' Ringer bicarbonate HEPES buffer (KRBH; 118 mMNaCl, 4.7 mM KCl, 1.1 mM KH₂PO₄, 25 mM NaHCO₃, 3.4 mM CaCl₂, 2.5 mMMgSO₄, 10 mM HEPES and 2 mg/ml BSA, pH 7.4) with 2.5 mM glucose for 90min at 37° C. KRBH buffer was then replaced with KRBH buffer containingeither 27.7 or 5.5 mM glucose for another 90 min at 37° C. Supernatantswere collected to determine insulin release. For serum insulindetection, 0.1 ml of tail-vein blood was collected from each mouse atthe end of 8 weeks after surgery, and serum was used for insulindetection. Insulin was measured by ELISA (Millipore, Billerica, Mass.).

Statistical Analysis

Data were presented as mean±SD. Differences were analyzed by Student'st-test. P<0.05 was considered statistically significant.

Example 2 Direct Reprogramming of Fibroblasts into DefinitiveEndoderm-Like Cells (DELCs)

This Example describes procedures for generating definitiveendoderm-like cells from mouse embryonic fibroblasts.

The doxycycline (Dox)-inducible secondary mouse embryonic fibroblast(MEF) system (Wernig, Lengner et al. 2008; Hanna, Saha et al. 2009) wasemployed to enable expression of the conventional four iPSC factors (theYamanaka factors or Oct4, Sox2, Klf4, and c-Myc) with precise temporalcontrol. MEFs were carefully prepared using standard procedures and usedfor reprogramming after 3-4 passages. Although endoderm cells may existin starting MEF populations, no such contamination of these cells wasobserved in the cultures employed.

To extend and test the iPSC-factor-based lineage-specific reprogrammingparadigm to endoderm, a two-step process was first devised to directlyreprogram secondary MEF cells into definitive endoderm-like cells(DELCs). The first step (step I) involved culturing secondary MEF cellsin media (Med-I) that is supplemented with 4 μg/ml Dox to initiateepigenetic activation. The second step (step II) was culturing theepigenetically activated cells in Med-II supplemented with 50 ng/mlActivin A and 1 mM LiCl (hereafter referred to as Activin/Li). Activin Aplus LiCl was used as the definitive endoderm induction conditionbecause chemical activation of the canonical WNT signaling pathway byLiCl synergized with Activin A-mediated Nodal signaling to promoteinduction of definitive endoderm cells from mouse ES cells (Li et al.2011). Sox17 and Foxa2, two relatively specific markers for definitiveendoderm (Tam, Kanai-Azuma et al. 2003; Qu, Pan et al. 2008), wereexamined by immunostaining at the end of Step II. By observing theresults of different durations of Step I and Step II, it was determinedthat 6-days of Step I followed by 6 days of Step II was an effectivecondition to generate Sox17⁺/Foxa2⁺ cells with relatively highefficiency. Consistent with results observed by the inventors in studieson cardiac and neural inductions using this paradigm, a shortened firststep induction of down to 4 days could still produce homogenousSox17/Foxa2 double positive colonies with fewer numbers, while prolongedfirst step induction, such as 7 days or 8 days, dramatically decreasedthe percentage of Sox17/Foxa2 double positive colonies. Hereafter theseSox17/Foxa2 double-positive cells are referred to as definitiveendoderm-like cells (DELCs).

In previous cardiac and neural reprogramming studies and the initialendoderm reprogramming using the iPSC-factor-based lineage-specificreprogramming paradigm, cell specification by lineage-specific signalswas only induced after initial epigenetic activation with iPSC factors.Therefore, whether lineage-specific signals could overlap withepigenetic activation to enhance the efficiency of reprogramming(towards a lineage-specific fate) is unclear.

The inventors hypothesized that transcriptional activity downstream oflineage-specific signals, in conjunction with iPSC factors that erasethe starting cell's epigenetic identity during early reprogramming,could help set up lineage-specific transcriptional programs. To testthis hypothesis, conditions were evaluated in which definitive endoderminduction factors (Activin/Li) were added to fibroblasts either after(Approach-I) or during (Approach II) iPSC factor expression, using Sox17and Foxa2 expression as a measure of DELC induction (FIG. 1A). Incontrast, addition of Activin/Li during the iPSC-factor expression(Approach-II) yielded mRNA levels of Sox17 and Foxa2 1.5 fold greaterthan strictly separating iPSC factor expression and Activin/Li inductionin the two steps (Approach-I) (FIG. 1B). Consistently, addition ofActivin/Li during the iPSC-factor expression (Approach-II) also greatlyincreased the percentage of Sox17/Foxa2 double positive colonies (FIG.1C-1D). By using the Approach-II, 6-days iPSC factor expression in thepresence of Activin/Li followed by Activin/Li treatment for another 6days, other definitive endoderm marker genes, including Cerberus 1 (Cer)and C—X—C chemokine receptor type 4 (Cxcr4), were highly induced inaddition to Sox17 and Foxa2 (FIGS. 2B-2C). These results supported thehypothesis that transcriptional activities downstream oflineage-specific signals could participate early to set up thelineage-specific transcriptional programs in conjunction with iPSCfactors.

To establish a proof of concept that these DELCs have thedifferentiation potential toward pancreatic lineages, the DELCs wereinitially subjected to published pancreatic differentiation conditions(D'Amour et al., 2006; Li et al., 2011). Notably, like definitiveendoderm cells derived from mouse embryonic stem (mES) cells, the DELCsgenerated by the methods described herein also gave rise to cells thatexpressed specific markers for pancreatic progenitors, includingpancreatic and duodenal homeobox gene-1 (Pdx1) (Offield, Jetton et al.1996), hepatocyte nuclear factor-6 (Hnf6) (Jacquemin, Durviaux et al.2000), paired-box gene 6 (Pax6) (Sander, Neubuser et al. 1997), and NKhomeobox factor 6.1 (Nkx6.1) (Sander, Sussel et al. 2000), as examinedby immunostaining and real-time PCR (FIG. 2D-2E). The cells expressingpancreatic progenitor markers are referred to herein as pancreaticprogenitor-like cells (PPLCs). Further differentiation of the PPLCs invitro could give rise to insulin expressing cells, as examined byimmunostaining and real-time PCR (FIG. 2F-2G). These results demonstratethat the DELCs generated as described herein could differentiate all theway to pancreatic β-like cells, like definitive endoderm cells derivedfrom mES cells.

However, the efficiency of generating pancreatic-like cells from theDELCs was low, although comparable to differentiation of mES cells usingthe same conditions. For example, only about 5% and 10% of cells werepositive for just Nkx6.1 or Pdx1, respectively, and only about 1% werePdx1/Nkx6.1 double positive. Further differentiation in vitro gave riseto less than 0.1% insulin expressing cells. And consistent with previousstudies (D'Amour et al., 2006), these insulin expressing cells didn'tresponse well to high glucose stimulation. These results indicate thatthe lineage induction conditions ideally should be further improved.Therefore, the next Example describes efforts for optimizing inductionconditions for generating pancreatic-like cells by small molecules.

Example 3

Identification of Novel Combinations of Small Molecules that EnhanceGeneration of Pancreatic Progenitor-Like Cells

This Example describes experiments demonstrating that several smallmolecules can be used to generate pancreatic progenitor-like cells fromcells that express factors such as Cerberus 1 (Cer), C—X—C chemokinereceptor type 4 (Cxcr4), Sox17, and/or Foxa2.

Beta cell formation and maturation are impaired in Nkx6.1-null mutantmice and can be restored upon re-expression of Nkx6.1 in multipotentialPdx1+ pancreatic progenitors (Nelson et al., 2007). In other studies,Pdx1+/Nkx6.1+ pancreatic progenitor cells differentiated from human EScells could be purified with the cell-surface marker CD142, and aftertransplantation into mice, these purified progenitor cells gave rise toall pancreatic cell lineages, including glucose-responsive,insulin-secreting cells (Kelly et al., 2011). These studies suggest thatdual expression of Pdx1 and Nkx6.1 is an indicator that pancreaticprogenitor cells can give rise to functional β cells. Induction of Pdx1⁺and Nkx6.1⁺ expression was used in experiments designed to identifyconditions that generate pancreatic progenitor-like cells (PPLCs) fromthe reprogrammed DELCs.

An available drug collection of 400 compounds in the 48-well format wasscreened to identity small molecules that induce high percentages ofPdx1⁺/Nkx6.1⁺ cells in DELC populations. Med-III medium (DMEMsupplemented with 1×B27, 2 mM Glutamax) was used as the basal conditionand double staining of Pdx1 and Nkx6.1 at 4 days post-induction was usedas the readout.

Induction of Pdx1 and Nkx6.1 expression by small molecules is referredherein to the “third step” (step III) of pancreatic induction. Severalprimary hits that could increase Pdx1 and/or Nkx6.1 expression werefurther characterized using larger-well cultures. Confirmed hitsincluded retinoic acid (RA, a RAR agonist), A83-01 (a TGFβ receptorinhibitor), 2-phospho-L-ascorbic acid (pVc), and LDE225 (a hedgehogpathway inhibitor) (FIG. 3). After testing various small moleculecombinations, significantly improved pancreatic induction was achievedby treating DELCs with 2 μM RA, 1 μM A83-01, 2 μM LDE225 and 280 μM pVcfor 1 day, and then 1 μM A83-01, 2 μM LDE225 and 280 μM pVc for another3 days. Under these conditions, the pancreatic progenitor makers Pdx1,Nkx6.1, Hnf6, Pax6 and Sox9 (Lynn, Smith et al. 2007) were highlyexpressed and co-localized as detected by co-immunostaining (FIG. 4B).About 35% of the cells expressed Pdx1 and about 30% of the cellsexpressed Nkx6.1, 2.5-fold and 5-fold increase, respectively, overprevious conditions (POC approach). More importantly, about 8% of thecells were Pdx1/Nkx6.1 double positive, a 7-fold increase over previousconditions (POC approach). qRT-PCR analysis further confirmed heightenedexpression of Pdx1, Nkx6.1, Pax6 and Hnf6 during the pancreaticinduction process (FIG. 4C). These results indicate that this novelcombination of small molecules promoted differentiation of DELCs intoPdx1⁺/Nkx6.1⁺ PPLCs.

The inventors then hypothesized that overlapping lineage-specificpatterning conditions based on small molecules with early reprogrammingwould enhance pancreatic induction. To this end, further screens of theinventors' drug collection were performed during the first- andsecond-step inductions, with double staining of Pdx1 and Nkx6.1 at theend of the third step (day 16) as a readout. Remarkably, several smallmolecules, when applied during the first and/or second steps, greatlyenhanced pancreatic induction. In particular, 1 μM Bix-01294 (a G9ahistone methyltransferase inhibitor), when added from DO to D6, and 280μM pVc, when added from DO to D12, increased the number of Pdx1+/Nkx6.1+colonies to about 3 fold and 4 fold respectively (FIG. 4D-4E). Furthertesting revealed that the combination of 1 μM Bix-01294 (added from DOto D6) and 280 μM pVc (added from DO to D12) generated about 8 fold ofPdx1+/Nkx6.1+ colonies (FIG. 4D-4E). Interestingly, treatment with Bixand/or pVc had no significant influence on the numbers of Sox17 andFoxa2 single positive and double positive cells.

Therefore, conditions and small molecules have been identified forefficiently generating pancreatic progenitor-like cells (PPLCs).

Example 4

Pancreatic Progenitor-Like Cells Can Be Further Differentiated intoMature Pancreatic-Like Cells

This Example describes experiments demonstrating that the pancreaticprogenitor-like cells generated as described above can be differentiatedinto more mature pancreatic-like cells.

The pancreatic progenitor-like cells generated as described herein weretreated with Med-IV medium containing laminin, nicotinamide, and B27etc. as previously reported (Schroeder et al., 2006) to determinewhether those PPLCs can be further differentiated into mature pancreaticlike cells. After 9 days of culture in this medium, a small population(about 0.5%) of insulin and C-peptide double-positive cells wasdetected.

To improve efficiency of pancreatic maturation, the inventors' drugcollection was further screened in a “fourth” step (step IV) to identifycompounds that increase the percentage of cells that express insulin andPdx1 on day 25. These experiments showed that 5 μM SB203580 and 280 μMpVc significantly increased the number of insulin+/Pdx1⁺ double-positivecells when used individually, and when combined together these compoundssynergistically enhanced the numbers of insulin⁺/Pdx1⁺ cells in thepopulation (e.g., to about 2%) (FIG. 5A).

The expression levels of insulin, Pdx1 and Nkx6.1 in the pancreaticprogenitor-like cells treated with SB203580 and pVc also increasedsignificantly (FIG. 5B). Glucagon-producing pancreatic alpha-like cells,somatostatin-producing pancreatic delta-like cells, andamylase-producing pancreatic acinar-like cells were also detected on day25 (FIG. 5C). Importantly, these endocrine-like cells produced only onehormone, a defining characteristic of mature pancreatic endocrine cells.The insulin-positive cells were also Nkx6.1-positive and secretedinsulin when stimulated with high level of glucose (FIG. 5C-5D). Thus,these differentiated PPLCs gave rise to pancreatic endocrine andexocrine-like cells in vitro, including functional insulin-secretingbeta-like cells.

Example 5

iPSCs Were Not Generated during the Reprogramming of Fibroblasts toPancreatic-Like Cells

This Example illustrates that the reprogramming procedure described inExample 2 does not generate fully pluripotent stem cells.

Generating iPSCs from secondary MEFs requires at least 9 days of Doxtreatment with LIF, followed by an additional 7-10 days of culture, asconfirmed by expression of a knock-in GFP reporter for Nanog, apluripotency gene (Wernig, Lengner et al. 2008). However, when theprocedures described herein were employed, no Nanog-GFP-positive cellswere detected during reprogramming of MEFs to DELCs, as assessed viatime-lapse imaging (FIG. 6B). This finding is consistent with theinventors' previous studies on direct cardiac, neural and endothelialcell reprogramming. As detected by qRT-PCR, Nanog and Rex1 (anotherpluripotency gene) expression remained nearly undetectable during thewhole process, while endoderm marker genes were induced from day 4 andpeaked on day 12 (FIG. 6A). On day 12, the expression levels of theseendoderm marker genes were comparable to or higher than those ofiPSC-derived definitive endoderm cells (FIG. 6A).

Similarly, Nanog-GFP-positive cells were not detected by time-lapseimaging during the third and fourth steps of differentiation (FIG.6C-6D). Expression of the pancreatic progenitor markers Pdx1/Nkx6.1 weredetected at the end of step III, and the endocrine pancreatic markersinsulin and Glucagon were detected at the end of step IV byimmunostaining and/or by q-RT-PCR (FIG. 6A, 6C, 6D). These resultsconfirmed that the methods described herein can directly reprogram MEFcells to DELCs and eventually generate pancreatic-like cells withoutstarting from iPSCs.

Example 6 In Vivo Characterizations of Pancreatic Progenitor-Like Cells

This Example shows that the pancreatic progenitor-like cells generatedas described above can give rise to insulin secreting pancreatic betacells that can regulate blood glucose levels in vivo.

To assess PPLC function in vivo, PPLCs were transplanted under thekidney capsules of immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ(NSG) mice, which had been induced to become hyperglycemic byintraperitoneal injection of STZ (Streptozocin). Mice were considereddiabetic when blood glucose measurements were >300 mg/dl for 4consecutive days, at which stage they were used as transplantrecipients. Each recipient received a renal subcapsular transplant ofabout 3×10⁶ pancreatic progenitor-like cells or 3×10⁶ MEF cells.Untreated normal and STZ-only-treated NSG mice were used as controls.Blood glucose levels were measured weekly after surgery.

In the untreated control group, blood glucose levels remained normal, asexpected (FIG. 7A). The lower line (X symbols) shows the blood glucoselevels of normal mice. As shown in FIG. 7A, the STZ-only group (filledtriangles), glucose levels increased gradually and peaked by the end ofthe third week. In the STZ-treated group that received MEF cells (filledsquares), glucose levels increased similar to the STZ-only group.

In contrast, STZ-treated mice, who had received transplantation ofPPLCs, exhibited a transient increase in glucose levels within the firstweek that decreased gradually thereafter and that approached levels innormal mice by about week 7 (FIG. 7A, filled diamonds). These dataindicate that the implanted PPLCs differentiated into insulin-secretingcells within the first 2 weeks of transplantation. At the end of 3weeks, the kidneys of three mice from each of the two cell-transplantedgroups were harvested for immunofluorescence analyses. In kidneycapsules engrafted with PPLCs, glucagon-producing pancreatic alpha-likecells, insulin-producing pancreatic beta-like cells,somatostatin-producing pancreatic delta-like cells, amylase-producingpancreatic acinar-like cells, and Pdx1⁺/Ck19⁺ pancreatic ductal-likecells were identified (FIG. 7B). In contrast, none of these genes wereexpressed in kidney capsules engrafted with MEF cells. Furthermore, thedetected insulin-positive cells were also Pdx1- and Nkx6.1-positive(FIG. 7C). Importantly, these endocrine-like cells were nearly allsingly hormonal, and bi-hormonal cells were rarely detected (FIG. 7C),which is consistent with the in vitro findings described herein.

To assess serum insulin levels, blood samples were collected at the endof 8 weeks and analyzed by ELISA. Transplantation of PPLCs to thediabetic mice significantly increased the level of circulating insulin,compared with MEF-grafted mice (FIG. 7D). There was no significantdifference in serum insulin levels between STZ-only mice and STZ micetransplanted with MEF cells. These results showed that PPLCs couldmature in vivo into cells of all three pancreatic lineages, includingfunctional, insulin-secreting beta cells that help to amelioratehyperglycemia in vivo.

Example 7

Materials and Methods for Conversion of Human Fibroblasts intoEndodermal Progenitor Cells

This example describes procedures for generating endodermal progenitorcells from human fibroblasts.

Conversion of Human Fibroblasts into Endodermal Progenitor Cells

All cell culture products were from Invitrogen/Gibco BRL and allchemicals and growth factors were from Stemgent except where mentioned.

Human foreskin fibroblasts (CRL-2097, ATCC) were cultured in a 10 cmtissue culture dish in regular fibroblast culture medium. Reprogrammingwith episomal vectors was done as described by Okita et al. (2011).Briefly, 4×10⁵ fibroblasts were electroporated with up to 6 μg ofepisomal vectors (pCXLE-hOCT3/4-shp53-F, pCXLE-hSK and pCXLE-EGFP) usingthe Microporator Human Dermal Fibroblast (NHDF) Nucleofector™ Kit(Lonza) according to the manufacturer's instructions. Cells werecultured in fibroblast medium for 4 days and then re-plated ontoMatrigel-coated 10 cm dishes at a density of 50,000 cells per dish. Thecells were then cultured in reprogramming initiation medium (DMEM/F12,10% Knockout serum replacement, 5% ES-FBS, 1% Glutamax, 1% Non-essentialamino acids, 1% penicillin/streptomycin, 0.1 mM β-mercaptoethanol, 10ng/ml bFGF, 10 ng/ml EGF, 2 μM Parnate, 0.5 μM RG108, 0.1 mM sodiumbutyrate, 0.5 μM NECA, and 3 μM CHIR99021) for 1 week, followed byendodermal induction medium (Advanced RPMI, 2% ES-FBS, 1% Glutamax, 1%Non-essential amino acids, 1% penicillin/streptomycin, 0.1 mMβ-mercaptoethanol, 2 μM Parnate, 0.5 μM RG108, 0.1 mM sodium butyrate,0.5 μM NECA, 3 μM CHIR99021, and 100 ng/ml Activin A) for another 2-3weeks. The converted colonies were carefully picked up for expansion inexpansion medium (DMEM, 1% Glutamax, 0.5×N2, 0.5×B27 media supplement(Invitrogen), 5 μg/ml BSA, 1% penicillin/streptomycin, 10 ng/ml bFGF,10n g/ml EGF, 0.5 μM A83-01, and 3 μM CHIR99021) and were passaged at1:4-1:6 each time by Accutase treatment. Routinely, 0.5 μM thiazovivinwas used during the first 12-24 hour period of each passage to preventcell death after dissociation.

Differentiation of Posterior Foregut-Like Progenitor Cells (cPF Cells)into Pancreatic Endodermal Progenitor Cells (cPE Cells)

For pancreatic differentiation, posterior foregut-like progenitor cellswere cultured in pancreatic differentiation medium (DMEM, 1% Glutamax,B27, 5 μg/ml BSA, 1% penicillin/streptomycin) with 25 ng/ml FGF7, 25ng/ml FGF10, 0.5 μM A83-01, 0.1 μM Compound E, 2 μM Retinoic acid, 0.1μM GDC-0449, and 0.1 μM LDN-193189 for 2 days; then 50 ng/ml EGF, 50ng/ml Exendin-4, 0.5 μM A83-01, 0.1 μM Compound E, 50 nM TPB, 0.1 μMLDN-193189, and 10 mM nicotinamide for another 3 days. Afterdifferentiation, the pancreatic endodermal progenitor (cPE) cellpopulation was passaged with Accutase and cultured in cPE expansionmedium (DMEM, 1% Glutamax, 1×B27, 5 μg/ml BSA, 1%penicillin/streptomycin, 10 ng/ml bFGF, 50 ng/ml EGF, and 0.5 μMA83-01). Routinely, 0.5 μM thiazovivin was used during the first 12-24hour period of each passage to prevent cell death after dissociation.

Maturation of Pancreatic Endodermal Progenitor (cPE) Cells intoPancreatic Beta-Like Cells (cPB Cells) In Vitro

The pancreatic endodermal progenitor (cPE cells) were differentiatedinto beta-like cells in the pancreatic maturation media with DMEM, 1%Glutamax, B27, 5 μg/ml BSA, and 1% penicillin/streptomycin, 50 ng/mlExendin-4, 1 μM A83-01, 10 μM forskolin, 10 μM dexamethasone, 10 mMnicotinamide, 0.1 μM Compound E, 50 μg/ml vitamin C, and 2 μM Bayk-8644for 10 d, and then cultured as 3D aggregates in low-attachment platesfor another 8-12 days.

Immunofluorescence Staining

Standard immune-staining was carried out as previously reported by Russet al. (PLoS One 6, e25566 (2011)). For cell cluster staining primaryand secondary antibodies were incubated overnight at 4° C. Secondaryantibodies were Alexa Fluor conjugated (1:500-1000) (Invitrogen). Nucleiwere visualized by Hoechst (Sigma-Aldrich) staining Images were capturedusing a Nikon Eclipse TE2000-U microscope or a SP5 confocal microscope.The primary antibodies used included SOX17, FOXA2, NANOG, HNF6, andHNF4A, with DAPI staining for nuclei. Representative images of cellsfrom at least five independent experiments are shown in FIGS. 8B, 8C,9G, and 9H. A representative image of at least three independentexperiments is shown in FIGS. 9C, 9L, 10B, 10C, 10G, and 11B. Arepresentative picture of three independent experiments is shown inFIGS. 8G, 11B, 11G and 11H. A representative image of two independentmice is shown in FIG. 10L.

Flow Cytometry

Cells were harvested at the indicated time points by Accutase treatment,fixed with 4% formaldehyde solution, and washed five times with ice-coldPerm/Wash buffer (BD). Cells were aliquoted and incubated individuallyor combinatorially with antibodies and isotype controls on ice for twohours. Cells were washed with Perm/Wash buffer for five times andincubated individually with Alexa Fluor 488-conjugated or Alexa Fluor555-conjugated antibodies (1:500, Invitrogen) on ice for one hour. Cellswere washed with Perm/Wash buffer for five times, re-suspended in 0.5 mlice-cold PBS with 2% FBS, and analyzed by FACSCalibur and CellQuestsoftware (BD). FlowJo software (Tree Star) was used to analyze the data.

Quantitative PCR

For quantitative PCR analysis, total RNA was extracted using the RNeasyPlus Mini Kit in combination with QIAshredder (Qiagen). First strandreverse transcription was performed with 1 μg RNA using iScript™ cDNASynthesis Kit (BioRad). Quantitative PCR was taken out using iQ SYBRGreen Supermix (Bio-Rad).

Kidney Capsule Transplantation

Mice used in this study were maintained according to protocols approvedby the University of California, San Francisco, Committee on LaboratoryAnimal Resource Center. The kidney capsule transplantation was done aspreviously reported⁴⁰. Briefly, cells were collected from culture dishesby cell scraper, and injected under the renal capsule ofimmune-deficient NSG (NOD.Cg-Prkdc^(scid) Il2rg^(tmlWjl)/SzJ) male mice(six to ten week old). For transplantation assays of established cPFcell lines, hESC-derived definitive endoderm generated bydifferentiation for 1 day in RPMI containing 100 ng/ml Activin A and 50ng/ml WNT3a, followed by 4 days in RPMI containing 0.2% FBS and 100ng/ml Activin A served as control. Grafts were dissected and analyzed atthe indicated time points.

Glucose Stimulated Insulin Secretion (GSIS) Assays

Cell were pre-incubated for 1 hour in Krebs-Ringer buffer (KRB),followed by incubation for 1 hour in KBR containing 2.8 mM glucosefollowed by 1 hour incubation in KRB containing 16.7 mM Glucose followedby 30 min in KRB containing 16.7 mM Glucose and 30 mM KCl HumanC-peptide levels were quantified using an ultrasensitive ELISA kit(Mercodia; cross-reactivity with insulin and pro-insulin, 0.0006% and1.8%, respectively).

Statistics.

Indicated P values were obtained using a two-tailed t-test, and allquantitative data are shown as mean±s.e.m. No statistical method wasused to predetermine sample size. No samples were excluded. Theexperiments were not randomized. The investigators were not blinded toallocation during the experiments and outcome assessment.

Example 8

Direct Conversion of Human Fibroblasts into Definitive EndodermalProgenitor Cells

Human foreskin fibroblasts were transduced with non-integrating episomalreprogramming factors, OCT4, SOX2, KLF4, and a short hairpin RNA againstp53 as described in Example 7. The cells were allowed to recover infibroblast medium for four days, and then they were cultured ininitiation medium containing epidermal growth factor (EGF), basicfibroblast growth factor (bFGF) and CHIR99021 (an activator of WNTsignaling) to support cell proliferation (FIG. 8A). After seven days,the culture conditions were switched to endodermal conversion mediacontaining CHIR99021 and high level of Activin A (100 ng/ml) toestablish converted definitive endodermal progenitor (cDE) cells, basedon previous studies demonstrating key roles for the Activin A and WNTsignaling pathways in endodermal fate decision in vitro and in vivo(Stainier et al. (2002); Tian et al. (2006); D'Amour et al. (2005)).This basic endodermal conversion protocol gave rise to cell colonieswith an epithelial morphology at day 21 to 28 (FIG. 8B). Coloniesspecifically stained for definitive endodermal progenitor markers SOX17and FOXA2, but not for the pluripotency marker NANOG or primitive gutmarker genes HNF4α and HNF6 (FIGS. 8C and 8G). As expected, controlfibroblasts were negative for endodermal and pluripotency markers (FIG.8G). To increase the efficiency of the endodermal conversion protocol, asmall scale screening was performed of chemical compounds known tocontain bioactive molecules capable of directing cell differentiation(Zhu et al. (2010). The addition of a combination of epigeneticmodulators, including sodium butyrate (NaB, a histone deacetylaseinhibitor), Parnate (Par, a histone demethylase inhibitor), and RG108(RG, a DNA methyltransferase inhibitor), significantly improved theconversion efficiency by 2.5 fold (FIG. 8D). Additional screening usingthis improved condition revealed that 5′-N-ethylcarboxamidoadenosine(NECA, an adenosine agonist) could further increase the conversion rateby 2-fold, resulting in 5-fold increase over the basal protocol (FIG.8D). Notably, by employing this optimized condition it was possible togenerate approximately fifty FOXA2 and SOX17 double-positive cDEcolonies from 400,000 human fibroblasts (FIG. 8D). Quantitativepolymerase chain reaction (qPCR) revealed a gradual up-regulation ofboth SOX17 and FOXA2 gene transcripts starting at day 14 (FIG. 8E). Incontrast, endogenous pluripotency gene transcripts NANOG and OCT4 wereundetectable during the whole conversion process. Additional flowcytometric analysis revealed a similar gradual increase in FOXA2⁺ cellsstarting at day 21, without detection of cells immune-reactive for thepluripotency marker TRA-1-60 at any stage (Chan et al. (2009) (FIG. 8F).These data indicate that human fibroblasts can be directly convertedinto definitive endodermal progenitor cells without transitioningthrough a pluripotency state. Thus, the protocol described hereinefficiently converts mesodermal human fibroblasts into endodermalprogenitors employing the Cell-Activation and Signaling-Directed (CASD)transdifferentiation approach.

Example 9 Specification, Expansion and Characterization of PosteriorForegut-Like Progenitor Cells

This Example describes experiments designed to expand definitiveendodermal progenitor cells by using a simple expansion media containingtwo small molecules, CHIR99021 (an activator of WNT signaling) andA83-01 (an inhibitor of TGFβ signaling) and two growth factors, EGF andbFGF that significantly promoted their expansion in serial passages(FIGS. 9A and 9B) Immunofluorescence analysis revealed strong expressionof endodermal progenitor markers SOX17 and FOXA2, but also induction ofprimitive gut tube marker HNF4a and posterior foregut marker HNF6 (FIG.9C), suggesting further specification towards posterior foregut-likeprogenitor cells (cPF cells). Employing these culture conditions,several posterior foregut-like progenitor cell lines were successfullyestablished from independent experiments that could be further expandedusing the same culture conditions (FIG. 9D). Posterior foregut-likeprogenitor cells proliferated rapidly with an average doubling time of 2days. After 15 passages, this represents at least a trillion-foldincrease in cell number (FIG. 9E). All four media supplements wereimportant for cPF cell self-renewal (FIG. 9F) and expanded posteriorforegut-like progenitor cells maintained their epithelial colonymorphology as well as posterior foregut-like phenotype as determined byimmunofluorescence staining for SOX17, FOXA2, HNF4a, HNF6, and SOX9(FIGS. 9G and 9H).

During mouse embryonic development, Pdx1 expression is first detected atembryonic day 8.5 and marks the endodermal region that will give rise tothe whole pancreas, as well as the common bile duct, distal stomach, andduodenal epithelium (Oliver-Krasinski & Stoffers (2008); Spence et al.(2009). Interestingly, PDX1 protein (FIG. 9H) was detected but not themore specific pancreatic endoderm (PE) marker NKX6.1 (data not shown).Consistently, qPCR analysis demonstrated the induced high levelexpression of multiple posterior foregut progenitor gene transcripts,including SOX17, FOXA2, HNF1A, HNF1B, HNF4A, HNF6, SOX9 and PDX1 inforegut-like progenitor cells when compared to parental fibroblasts(FIG. 9I; FIG. 9O-9P). In contrast, ectodermal marker gene SOX1,mesodermal marker gene BRACHYURY, and pluripotency marker genes OCT4 andNANOG were not induced (FIG. 9I). Collectively, these data confirm thespecific posterior foregut identity of cPF cells.

The specific posterior foregut identity of cPF cells is furthersupported by the observations that cPF cells at both early and latepassages possessed comparable capacities to differentiate towards thehepatic lineage (FIG. 9M). In contrast, cPF cells rarely gave rise toNKX2.1 positive or CDX2 positive cells that would suggest specificationtoward the lung (anterior fore gut endoderm) and gut (midgut endoderm)lineages, respectively (date not shown). Of note, cPF cells can befrozen and thawed with high recovery rate, an important advantage formany downstream assays and uses.

The genome stability of cPF cells was also evaluated using comparativegenomic hybridization (CGH) arrays that did not demonstrate any grosschromosomal aberrations (data not shown). However, four copy numbervariants (CNVs) were detected in one human neonatal fibroblast-derivedcPF cell line, as shown in Table 3.

TABLE 3 Copy number variation Sample CNV Location Type AnnotationsCRL-2097, 8 Chr2: 89185302- Amp N/A p6 89301214 Chr4: 69392576- DelUGT2B17, UGT2B15 69483277 Chr6: 259316- Del DUSP22 362290 Chr8:39258894- Del ADAM5P, ADAM3A 39381514 Chr12: 9637323- Del N/A 9693948Chr14: 106405703- Amp ADAM6, 106803307 NCRNA00226 Chr14: 106803307- AmpNCRNA00221 107214893 Chr15: 22318597- Del LOC727924, OR4M2, 22558756OR4N4 cPF 12 Chr2: 89185302- Amp N/A (CRL-2097), 89301214 p17 Chr4:69392576- Del UGT2817, UGT2815 69483277 Chr6: 259316- Del DUSP22 362290Chr6: 78979161- Del N/A 79023328 Chr8: 39258894- Del ADAM5P, ADAM3A39381514 Chr11: 55385617- Del OR4P4, OR4S2, 55450788 OR4C6 Chr12:9637323- Del N/A 9693948 Chr14: 19794577- Amp POTEM, OR11H2, 204216770R4Q3 Chr14: 106405703- Amp ADAM6, 106803307 NCRNA00226 Chr14:106803307- Amp NCRNA00221 107214893 Chr15: 22318597- Del LOC727924,OR4M2, 22558756 OR4N4 Chr15: 34735949- Del N/A 34785082Copy number variation analysis revealed a total of 8 and 12 CNVs inneonatal fibroblasts and expanded cPF cells, respectively. The four newCNVs in expanded cPF cells are highlighted in Table 3 in bold withunderlining.

These results are consistent with a recent report demonstrating thatlong-term cultures of bipotent stem cells from adult human liver aregenomically-stable (Huch et al., Cell 160: 299-312 (2015)). Notably, theepisomal reprogramming vectors were spontaneously lost in establishedcPF cell lines (FIG. 9J), thus overcoming a current safety concernassociated with the integration of viral vector basedreprogramming/conversion approaches. Another potential safety concernregarding cells with proliferative and stem cell capacity is theirpotential for tumor formation (Hentze et al., Stem Cell Res 2, 198-210(2009)). Transplantation of expanded cPF cells under the kidney capsuleof immune deficient mice did not result in tumor formation even afterprolonged periods up to 24 weeks in vivo (n=10) (FIG. 9K). In contrast,all controls (hESC-derived endoderm progenitor cell populations) formedtumorigenic structures with big cysts and increased graft size sevenweeks after transplantation (n=4) (FIG. 9K).

Immunofluorescence analysis of the cPF grafts demonstrated thatepithelial structures (FIG. 9N) express characteristic endoderm markers,including E-cadherin, HNF4a, PDX1, SOX9, and cytokeratin (FIG. 9L).These results demonstrate that cPF cells can be greatly expanded inculture while maintaining their posterior foregut endodermal phenotype.

Example 10

Differentiation of cPF Cells into Expandable Pancreatic EndodermalProgenitor Cells with the Ability to Mature into Functional Beta-LikeCells In Vivo

Recently, several studies have reported the use of small molecules andgrowth factors to achieve differentiation of hESC-derived primitive guttube and posterior foregut endoderm into pancreatic endoderm (Kroon etal. (2008); Rezania et al. (2012), Nostro et al. (2011); Kunisada et al.(2012)). Using a similar approach, different combinations of smallmolecules and growth factors were screened for differentiation ofposterior foregut-like progenitor cells (cPF cells) into more committedpancreatic endodermal progenitor cells (cPE cells). A two-step protocolwas optimized in which cPF cells were first exposed to FGF7, FGF10,A83-01, Compound-E (an inhibitor of Notch signaling), retinoic acid(RA), GDC-0449 (an antagonist of Sonic hedgehog), and LDN-193189 (aninhibitor of BMP4 signaling) for 2 days (FIG. 10A). Subsequently,differentiating cells were treated with EGF, Exendin-4 (an agonist ofglucagon-like peptide-1), A83-01, LDN-193189, phorbol 12,13-dibutyrate(PBDu; an activator of protein kinase C), Compound-E, and Nicotinamide(an inhibitor of polyADP-ribose synthetase) for another 3 days (FIG.10A). Resulting cPE cells continued to express high levels of FOXA2,HNF6, SOX9, and PDX1, as is expected for PE progenitor cells (Seymour &Sander (2011); Pan & Wright (2011)) (FIG. 10B). Most importantly, thistreatment resulted in the generation of PDX1 and NKX6.1 double-positivecells (FIG. 10C). NKX6.1 expression in common PDX1⁺ pancreaticprogenitors (before NKX6.1 expression becomes further restricted to betacells) marks their commitment to a more specificendocrine-/ductal-bi-potent progenitor cell type (Henseleit et al.(2005)). Notably, hESC-derived NKX6.1 and PDX1 double-positive PEprogenitor cells have been shown to be able to give rise to functionalbeta cells after transplantation (Kelly et al. (2011)). FACS analysisrevealed approximately 78% of PDX1 positive cells, and 17.3% PDX1 andNKX6.1 double-positive cells within cPE populations at passage 1 (FIG.10D).

Next, the effect of cPF expansion media was tested on cPE cultures in anattempt to expand posterior foregut-like progenitor cells (cPE cells) ina similar fashion. However, cPE colonies easily detached from the plateunder the cPF expansion conditions. Omission of CHIR99021 and increasingthe EGF concentration to 50 ng/ml reversed this effect and promoted cPEcell expansion pursuant to the method illustrated in FIG. 10E. Underthis optimized culture condition, cPE cells were expanded more than twohundred million fold with an approximate doubling time of 3 days for upto 14 passages (FIG. 10F). Of note, expanded cPE cells maintained theirbi-potent progenitor identity as evidenced by the presence of PDX1 andNKX6.1 double-positive cells at passage 12 (FIGS. 10G and 10H).Consistently, qPCR results demonstrated the down-regulation of earlyendodermal marker gene SOX17. The pan-endodermal marker genes FOXA2 andHNF4A, as well as many pancreatic and endocrine marker genes, includingHNF6, PTF1A, HLXB9, and NGN3, were up regulated (FIG. 10I). More robustexpression was noted for other critical markers also highly expressed inmature beta cells, including PDX1, NKX2.2, and NKX6.1.

To explore whether expanded pancreatic endodermal progenitor cells (cPEcells) can further mature into functional beta cells in vivo, cPE cellswere transplanted under the kidney capsule of immune deficient mice.After 15-16, 19-20 and 23-24 weeks, human C-peptide was detected after aglucose challenge in the blood of 62.5%, 75%, and 86.6% of miceanalyzed, respectively, albeit at low levels (FIG. 10J). In addition, a2.1 fold increase in human C-peptide was detected in the serum of micebearing 23-week old grafts after glucose challenge when compared tofasting levels, a finding illustrating that cPE grafts become functionalupon transplantation into host animals (FIG. 10K). Starting at 15 weekspost transplantation, insulin-expressing beta-like cells and PDX1positive pancreatic progenitor cells were found in some graft sections,while other regions were negative for these and other pancreas markers(FIG. 10L). Insulin-positive cells co-expressed the critical beta celltranscription factors NKX6.1 and PDX1, but did not show expression ofother endocrine hormones (FIG. 10L). In summary, the data providedherein demonstrates that cPF cells can be differentiated into cPE cells,which can be greatly expanded in vitro while maintaining their specificphenotype. Importantly, cPE cells differentiate further in vivo towardsinsulin-producing, single-hormonal cells capable of releasing Insulin inresponse to glucose challenge.

Example 11

Maturation of cPE Cells into Insulin-Producing, Glucose-ResponsivePancreatic Beta-Like Cells In Vitro

Full maturation towards glucose responsive cells is optimallyillustrated by transplantation of in vitro differentiated pancreasendoderm progenitor cells into immune compromised mice. To address thequestion of whether the cPE cells generated as described herein candevelop into functional beta-like cells under cell culture conditions,cPE cells were incubated in a basal pancreatic differentiation mediathat has been shown to promote hESC/iPSC-derived pancreatic progenitordifferentiation into insulin-producing cells (Kunisada et al. (2012)).This media includes A83-01, Nicotinamide, Forskolin (an activator ofadenylyl cyclase), Dexamethasone (an agonist of glucocorticoidreceptor), and Exendin-4 (FIG. 11A). While C-peptide positive cellsexpressing high levels of PDX1 were consistently observed after 10-14days in culture (FIG. 11B), the relatively low number of cells (˜0.5%)indicated further optimization of conditions may improve the frequencyof conversion to functional beta-like cells. Chemical compounds able topromote differentiation of definitive endoderm (Borowiak et al. (2009))and PDX1⁺ progenitors (Chen et al. (2009) have been identifiedpreviously. However, molecules directing the final steps ofdifferentiation into beta-like cells have not been uncovered, likely dueto difficulties in generating and maintaining sufficient numbers ofdifferentiation-competent pancreatic endodermal progenitor cells.

A chemical screen was performed with the intent of identifying factorsthat would result in the more efficient development of C-peptidepositive cells from expanded cPE cells. These experiments revealed thatsupplementation of the basal pancreatic differentiation mediaindividually with Compound-E (an inhibitor of Notch signaling), VitaminC, or BayK-8644 (a Ca²⁺ channel agonist), were effective in increasingthe percentage of C-peptide positive cells (FIG. 11C). Combinedtreatment with these compounds had an additive effect (Exendin-4,A83-01, Nicotinamide, Forskolin, Dexamethasone, Compound-E, Vitamin C,and Bayk-8644), resulting in the formation of up to 7% C-peptidepositive cells (FIG. 11C).

Insulin-producing cells generated from ESCs/iPSCs under publishedculture conditions are mostly immature as evidenced by co-expression ofendocrine hormones, lack of crucial beta cell transcription factors andthe absence of insulin secretion in response to physiological levels ofglucose (Nostro & Keller (2012)). The maturation defects may be partlydue to the lack of 3D organization normally present in islets ofLangerhans (Sasai (2013)).

Hence, free floating cell aggregates of cPE cells were generated thathad first been incubated as 2D cultures for 10 days in the improvedmaturation media. The majority of cells within the aggregates expressedthe pan-pancreas marker PDX1 after 8-12 days of 3D culture (FIGS. 11D,11F and 11G). In addition, many insulin-expressing cells co-expressingkey beta cell transcription factors including NKX6.1, NKX2.2, and NeuroDwere detected (FIGS. 11D and 11G). In contrast, insulin-expressing cellsonly rarely co-stained for the endocrine progenitor marker NGN3 or otherendocrine hormones, including Glucagon (GCG) and Somatostatin (SST)(FIGS. 11D and 11G). Of note, all insulin-positive cells also stainedfor a human specific C-peptide antibody, thus excluding possible insulinuptake from the media (FIG. 11H).

Considering the expression of key beta cell markers, these cells weredesignated converted pancreatic beta-like cells (cPB cells). One of thedistinguishing hallmarks of pancreatic beta cells is the ability torelease insulin upon glucose stimulation. Importantly, glucosestimulated insulin secretion (GSIS) assays demonstrated that in vitrogenerated cPB cells released insulin (as detected by human C-peptideexpression) in response to physiological levels of glucose (n=7, foldincrease 2.1±1.3, range 0.9-4.8) (FIG. 11E). Collectively, thesefindings demonstrate that the in vitro differentiation protocoldescribed herein directed conversion of human fibroblasts into beta-likecells that respond to physiological levels of glucose.

Example 12

Improved Method for Generating Pancreatic Beta-Like Cells fromPancreatic Endodermal Progenitor Cells

This Example describes improved methods for efficiently generating largenumbers of pancreatic beta-like cells that remain functional aftertransplantation as demonstrated by significantly higher levels ofcirculating human C-peptide in the serum of glucose-challenged mice whencompared to fasted controls.

Materials and Methods

Pancreatic endodermal progenitor (cPE) cells were differentiated intopancreatic beta-like cells (cPB) by successive exposure to differentmedia using a process referred to herein as Protocol 2 (FIG. 12A). In afirst step, pancreatic endodermal progenitor cells were cultured in thepancreatic maturation media containing DMEM, 1% Glutamax, B27, 5 μg/mlBSA, and 1% penicillin/streptomycin, 10 μg/ml Heparin, 10 μM ZnSO4, 10μM Alk5 inhibitor II (Enzo, Farmingdale, N.Y.); 0.1 μM LDN-193189, 1 μMT3, 0.1 μM Compound E, 2 μM BayK-8644, 0.25 mM vitamin C for seven days.

In a second step, the cells were cultured in media containing DMEM, 1%Glutamax, B27, 5 μg/ml BSA, and 1% penicillin/streptomycin, 10 μg/mlHeparin, 10 μM ZnSO4, 10 μM Alk5 inhibitor II, 1 μM T3, 1 mM N-cysteine,10 μM Trolox, 2 μM R428, 2 μM BayK-8644, 0.25 mM Vitamin C for anotherseven days.

The cells were then cultured as 3D aggregates in low-attachment platesfor another seven days.

Results

In addition to the Notch inhibitor that had already been employed in themethods described above, Vitamin C and BayK-8644 were added, and asshown in FIG. 12B, both compounds promoted expression of the INSULINgene. Vitamin C and BayK-8644 also increased the number of C-peptidepositive cells that co-expressed key beta cell transcription factorsPDX1 and NKX6.1 (FIG. 12C). C-peptide positive cells rarely co-stainedfor other endocrine hormones, including Glucagon (GCG) and Somatostatin(SST) (FIG. 12C). Quantitative

FACS analysis revealed the formation of about 15% monohormonalC-pep+GCG-SSTcells, with only few polyhormonal cells (FIGS. 12D and12E).

To identify cells with Insulin gene expression, cPE cells were infectedwith a lentivirus containing a minimal Insulin promoter driving afluorescence reporter expression (FIG. 12G) and the cPB cells generatedtherefrom were FACS sorted at 21 days after introduction of thelentiviral reporter construct and conversion via Protocol 2. FIG. 12Hshows the results of a qPCR analysis of the sorted cPB cells for theexpression of beta cell marker genes, including INS, PDX1, NKX6.1,NKX2.2, NEUROD1, PAX6, RFX6, MAFA, GCK, PCSK1, KIR6.2, SUR1, UCN3 andSLC30A8 in comparison to human islets. As shown, while a few genesexhibited slightly reduced levels of expression, the cPB cells generatedby Protocol 2 had levels of expression for most genes that werecomparable to those observed for human islets.

Glucose stimulated insulin secretion (GSIS) assays conducted with invitro generated cPB cells revealed a robust release of insulin inresponse to physiological levels of glucose (n=7, fold increase=2.0)(FIG. 12F). The stimulation index, as calculated by the ratio of insulinsecreted in high glucose to low glucose, was similar among the cPBcells, hESC-derived beta-like cells, and primary human islets. Thus,protocol 2 efficiently generates functional pancreatic beta-like cellsin vitro.

These in vitro generated cPBs were then evaluate for in vivo functioningby transplantation into immunodeficient mice (FIG. 13A). Importantly,while control human fibroblasts did not evoke any response, significantglucose stimulated insulin secretion was detected in cPB-transplantedmice two months after transplantation (FIG. 13B) Immunofluorescenceanalysis of cPB grafts revealed the presence of many islet-likestructures containing Insulin-expressing pancreatic beta-like cells.C-peptide positive cells co-express critical beta cell transcriptionfactors PDX1 and NKX6.1, but did not exhibit expression of otherendocrine hormones GCG and SST (FIG. 13C). Specific ablation of theendogenous beta cells by the toxin Streptozotocin (STZ) resulted inrapid onset of overt diabetes in all control animals, while cPB bearingmice remained euglycemic (FIG. 13D).

After 5 weeks post-STZ treatment cPB grafts were removed by unilateralnephrectomy. Nephrectomized mice rapidly developed diabetes,demonstrating that human fibroblast-derived cPB cells controlled glucoselevels in STZ treated mice. In summary, these results directlydemonstrate that cPB cells transplanted into surrogate mice arefunctional in vivo and protect mice from diabetes.

Moreover, committed pancreatic endodermal progenitor cells (cPE) cellsgenerated from adult human fibroblasts were differentiated intopancreatic beta-like cells at efficiencies comparable to neonatalderived cPB cells, with only few double hormone positive cells (FIGS.14A, 14B and 14C). In addition, human adult fibroblasts derived cPBcells are functional as demonstrated by their glucose stimulated insulinsecretion (FIG. 14D).

Example 13 Materials and Methods

Undifferentiated MEL1 INS^(GFP/W) reporter cells (Micallef et al, 2012)were maintained on mouse embryo fibroblast feeder layers (Millipore) inhESC media as described (Guo et al, 2013b). Suspension-baseddifferentiations were carried out as follows. Briefly, confluentcultures were dissociated into single cell suspension by incubation withTrypLE (Gibco). Cells were counted and each well of 6-well low-adherenceplates were seeded with 5.5×10⁶ cells in 5.5 ml hES media supplementedwith 10 ng/ml Activin A (R&D systems) and 10 ng/ml HeregulinB1(Peprotech). Plates were placed on an orbital shaker at 100 rpm toinduce sphere formation, as described (Schulz et al, 2012). To inducedefinitive endoderm differentiation, aggregates were collected 24 hourslater in a 50 ml falcon tube, allowed to settle by gravity, washed oncewith PBS and re-suspended in d1 media (RPMI (Gibco) containing 0.2% FBS,1:5000 ITS (Gibco), 100 ng/ml Activin A, and 50 ng/ml WNT3a (R&Dsystems)). Clusters from 3 wells were combined into 2 wells at thispoint and distributed into fresh low-attachment plates in 5.5 ml d1media. Media thereafter was changed daily, by removing either 4.5 mlmedia (at the end of d1) or 5.5 ml media the following days and addingback 5.5 ml fresh media until day 9. After day 9, only 5 ml of media wasremoved and added daily.

Differentiation employing published protocols has been described (Schulzet al, 2012; Rezania et al, 2012). Media in a simplified differentiationprotocol described herein includes or consists of, d2: RPMI containing0.2% FBS, 1:2000 ITS, and 100 ng/ml Activin A; d3: RPMI containing 0.2%FBS, 1:1000 ITS, 2.5 μM TGFbi IV (CalBioChem), and 25 ng/ml KGF (R&Dsystems); d4-5: RPMI containing 0.4% FBS, 1:1000 ITS, and 25 ng/ml KGF.d6-7: DMEM (Gibco) with 25 mM Glucose containing 1:100 B27 (Gibco), 3 nMTTNBP (Sigma); d8: DMEM with 25 mM Glucose containing 1:100 B27, 3 nMTTNBP, and 50 ng/ml EGF (R&D systems); d9: DMEM with 25 mM Glucosecontaining 1:100 B27, 50 ng/ml EGF, and 50 ng/ml KGF. d10-14: DMEM with25 mM Glucose containing 1:100 B27, 500 nM LDN-193189 (Stemgent), 30 nMTATA-Binding Protein (TBP; Millipore), 1000 nM Alki II (Axxora), and 25ng/ml KGF; d15-21: DMEM with 2.8 mM Glucose containing 1:100 GlutaMAX™(Gibco) and 1:100 NEAA (Gibco). Human islets were obtained from ProdoLaboratories or the UCSF Islets and Cellular Production Facility.

Mice

NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (NSG) were obtained from JacksonLaboratories. Mice used in this study were maintained according toprotocols approved by the University of California, San FranciscoCommittee on Laboratory Animal Resource Center. For kidney capsulegrafts, approximately 5.0×10⁶ hESC differentiated cells in spheres and4000 human islet equivalents were transplanted as described (Russ &Efrat, 2011; Szot et al, 2007). For glucose-induced insulin secretion,mice were fasted overnight and serum was collected before and afterintraperitoneal administration of 3 g/kg D-glucose solution. Forinduction of diabetes, mice were administered 35 mg/kg streptozotocinvia intraperitoneal injection for 5 days. Graft bearing kidneys wereremoved for immunofluorescence analysis. No statistical method wasemployed to determine sample size, mice were not randomized and analysiswas not blinded.

Cell Sorting and Flow Cytometric Analysis

Briefly, spheres were collected and allowed to settle by gravity.Clusters were washed once in PBS and dissociated by gentle pipettingafter 12-15 minutes incubation in Accumax (Innovative CellTechnologies). For sorting, cell suspensions were filtered andre-suspended in FACS buffer consisting of phosphate-buffered saline(PBS) (UCSF cell culture facility) containing 2 mM EDTA (Ambion) and 1%BSA (Sigma). Dead cells were excluded by DAPI (Sigma) staining Cellsorting was performed on a FACS Aria II (BD Bioscience). For flow-basedanalysis, dissociated cells were fixed with 4% paraformaldehyde(Electron Microscopy Science) for 15 minutes at room temperature,followed by two washes in PBS. Samples were either stored at 4 C orimmediately stained with directly conjugated antibodies. Data analysiswas performed with FlowJo software. Mouse Glucagon and mouse humanC-peptide antibodies were conjugated in-house by the UCSF Antibody Coreand/or with Antibody Labeling Kits (Molecular Probes) according tomanufacturer's instructions. Commercially available directly conjugatedantibodies, i.e., antibodies Human PAX6-Alexa647, Islet-1-PE,NKX6.1-Alexa647, NKX6.1-PE, Chromogranin A-PE, NeuroD1-Alexa647,PDX1-PE, and Ki67-Alexa647, were from BD Bioscience.

Electron Microscopic Analysis

Spheres were fixed by adding 37° C. 0.1M sodium cacodylate solution(Sigma) containing 2% paraformaldehyde (Electron Microscopy Science) and2.5% glutaraldehyde (Electron Microscopy Science), 3 mM CaCl₂ (Sigma),final pH 7.4. Spheres were then transferred to 4° C. for approximately18 hours, followed by standard processing and analysis by the ElectronMicroscope Lab/Diabetes Center Microscope Core.

Immunofluorescence Analysis

Spheres were fixed for 15-30 minutes at room temperature with 4%paraformaldehyde, followed by multiple washes in PBS. Whole mountstaining was performed in suspension, by first blocking overnight at 4°C. in blocking buffer consisting of CAS-block (Invitrogen) with 0.2%TritonX (Fisher). Primary antibodies were incubated overnight at 4° C.in blocking buffer, followed by washes in PBS containing 0.1% Tween-20(PBS-T, Sigma) and incubation in appropriate secondary antibodiesdiluted in PBS-T overnight at 4° C. The next day, clusters were washedin PBS-T followed by PBS and mounted with Vectashield (Vector) on glassslides. For sectioning of clusters, spheres were embedded in 2% Agar(Sigma), followed by dehydration, paraffin embedding, and sectioning.Cut sections were rehydrated and treated with an antigen retrievalsolution (Biogenex) before incubation with primary antibodies overnightat 4° C. in blocking buffer. The next day, sections were washed 3 timesin PBS-T and incubated with appropriate secondary antibodies for 30-40minutes at room temperature in PBS-T. Appropriate Alexa-conjugatedsecondary antibodies were purchased from JAX or Molecular Probes andused at 1:500 dilutions. Slides were washed in PBS-T and PBS beforemounting in Vectashield. Nuclei were visualized with DAPI. Images wereacquired using a Leica SP5 microscope or a Zeiss ApoTome. Primaryantibodies were employed as indicated in TABLE 4.

TABLE 4 Antigen Species Dilution Manufacturer Human C-peptide Mouse1:200 Chemicon Human C-peptide Rat  1:1000 DSHB Insulin Mouse  1:1000Sigma Insulin Guinea pig 1:500 DAKO Glucagon Mouse  1:1000 Sigma NKX6.1Mouse 1:100 DSHB NKX2.2 Mouse 1:20  DSHB PDX1 Goat 1:200 R&D systemsHuman NEUROG3 Sheep 1:300 R&D systems Ki67 Rabbit 1:100 NovoCastraqPCR Analysis

Total RNA was isolated with TRIZOL (Sigma) or micro/mini RNAeasy kit(Qiagen) and reverse transcribed using the iScript cDNA Kit (Bio-Rad)according to manufacturer's instructions. qPCR analysis was performed onan ABI 7900 HT Fast Real-Time PCR System (Applied Biosystems) and CFXConnect Real Time System (Biorad) using standard protocols. Primers wereTaqman Probes (Applied Biosystems) and/or as published previously (Liuet al, 2014). P-values were calculated using a two-tailed student'st-test.

Content Analysis

Insulin, human C-peptide and proinsulin analyses were performed bymeasuring an aliquot of acidic ethanol lysed clusters with commerciallyavailable ELISA kits (Insulin Cat. 80-INSMR-CH10, human C-peptide cat.80-CPTHU-CH01, and proinsulin Cat. 80-PINHUT-CH01; all from Alpco).Total DNA was quantified by PicoGreen (Invitrogen) assay and normalizedto the percentage of C-peptide-positive cells in each sample.

Western Blotting for Proinsulin/Insulin

Cell lysates were resolved on 4-12% acrylamide gradient SDS-PAGE gels(NuPAGE, Invitrogen) normalized to cellular DNA (Quant-iT dsDNA,Molecular Probes). The samples were then electrotransferred tonitrocellulose membranes and immunoblotted with guinea pig anti-insulin,which recognizes both proinsulin and insulin, as previously described(Haataja et al, 2013) Immunoblotting with anti-tubulin was used as aconfirmatory loading control. HRP-conjugated secondary antibodies(Jackson ImmunoResearch) were used for enhanced chemiluminescencedetection (Millipore). The analysis was performed four times withisolated human islets used as a positive control.

Glucose Stimulated Insulin Secretion

Human islets or hES-derived spheres were transferred into tubes andwashed twice with Krebs-Ringer Bicarbonate buffer (KRB) containing 2.8mM Glucose. Samples were incubated for one hour in 2.8 mM glucosecontaining KRB to allow equilibration of cells. The 2.8 mM buffer wasremoved and replaced with fresh KRB containing 2.8 mM glucose for onehour followed by incubation for another hour in KRB containing 16.7 mMglucose. After the incubation period, buffers were collected for humanC-peptide-specific ELISA analysis using a commercially available kit(Alpco).

Example 14

Pancreatic Differentiation of hESCs Using a Large-Scale Culture SystemResults in Two Distinct Subsets of Insulin-Producing Cells

To generate pancreatic beta cells from human PSC, a scalablethree-dimensional suspension culture system was developed that builtupon previously reported methods (Schulz et al, 2012; Rezania et al,2012) (FIG. 15A). To monitor the generation of live insulin-producingcells and facilitate their isolation, the recently published INS^(GFP/W)reporter cell line (Micallef et al, 2012) was utilized in which greenfluorescence protein (GFP) expression is under the control of theendogenous insulin promoter. Using this differentiation protocol, GFPreporter expression was readily observed at day 13 and increasedthereafter, resulting in an average of 37±8% GFP positive cells betweendays 19-24 (FIG. 15B-D). The validity of GFP as an accurate substitutefor insulin was verified by staining with an insulin-specific antibody,which revealed an even higher percentage of insulin-producing cells (upto 60%) likely due to delayed accumulation of the fluorescence marker(FIG. 15E). Similar results were obtained with an antibody specific tohuman C-peptide, excluding antibody reactivity due to insulin uptakefrom culture media (FIG. 15E). Co-staining for human C-peptide andglucagon (GCG), a hormone normally produced by alpha cells, showed that4.3% and 13.2% of all cells exhibited a polyhormonal phenotype at day 13and day 19, respectively (FIG. 15F). Co-staining for C-peptide andNKX6.1 at day 20 indicated the presence of some double positivepancreatic beta cells (FIG. 15G). Quantitative flow cytometry analysisrevealed that the proportion of INSULIN and NKX6.1 double-positivepancreatic beta cells increased from less than 2.5% at day 13 toapproximately 12% cells at day 19 of total cells (FIG. 15G).Ultrastructural analysis of differentiated cultures showed cellscontaining secretory vesicles with an electron dense core surrounded byan electron light halo (FIG. 15H), a morphology reminiscent of insulinvesicles that are found in human pancreatic beta cells. The majority ofcells, however, exhibited a mixture of secretory granules usually foundin non-beta cells of human pancreas preparations (FIG. 15H). Thus,differentiation experiments employing published protocols (Schulz et al,2012; Rezania et al, 2012) result in the efficient generation of twodistinct insulin-producing cell populations: INS⁺ cells that do notco-express the critical TF NKX6.1 and manifest as polyhormonal cells,and INS⁺/NKX6.1⁺ beta cells that more closely resemble human pancreaticbeta cells. Notably, INS⁺/NKX6.1⁺ beta cells are absent from cultures atearlier time points but appear and increase in number at later stages ofdifferentiation, indicating that they are derived from a distinctprogenitor cell type.

Example 15

Defining the Temporal Activities of Individual Signaling Factors toEfficiently Generate PDX1+ and PDX1+/NKX6.1+ Pancreas ProgenitorPopulations while Preventing Precocious Induction of EndocrineDifferentiation

To characterize the type of progenitors present in differentiatingcultures at the point of endocrine induction, a detailed time courseanalysis was performed for the expression of pancreatic markers PDX1,NKX6.1, NEUROG3, GCG and INS (FIG. 16). High expression of theprogenitor marker PDX1 was efficiently induced and maintained startingone day after the combined addition of Retinoic Acid (R), the SHHinhibitor Cyclopamine (C), and the BMP inhibitor Noggin (N) to theculture media (referred to as RCN, Day 6, FIGS. 16A and B). Subsequenttreatment with epidermal growth factor (EGF), KGF and N (EKN) resultedin the robust generation of PDX1⁺/NKX6.1⁺ double positive cells reaching67% of the total population at day 11 (FIGS. 16A and B).Immunofluorescence analysis revealed that the RCN cocktail of factorswidely used to generate pancreatic endoderm also induces precociousexpression of NEUROG3 in PDX1⁺ pancreatic progenitors. Indeed, theexpression of NEUROG3 can be detected as early as day 6, when NKX6.1protein is absent from all cells (FIGS. 16A and B). Consequentially,insulin-expressing cells that are first detected 4 days after NEUROG3induction (starting at day 10), do not co-express NKX6.1 and are mostlypolyhormonal (FIGS. 15F and G, and FIG. 16C). In contrast, INS⁺/NKX6.1⁺double positive pancreatic beta cells can be readily detected only atlater time points (day 19, FIG. 15G), indicating that these cellsdifferentiate from PDX1/NKX6.1 double positive progenitor cells. It wasthus expected that robust generation of PDX1⁺/NKX6.1⁺ progenitor cellsprior to induction of NEUROG3 would allow efficient generation ofpancreatic beta cells in vitro. To determine which of the factors usedbetween days 6-8 in the original protocol (R, C, and N) were responsiblefor the induction of PDX1, NKX6.1 and NEUROG3, spheres were incubatedwith each of the factors alone or in different combinations over days6-8 (FIG. 17A). Basal media with B27 but lacking any additional factorsserved as the control condition. At the end of day 8, each of these sixconditions was further subdivided into three different treatment groups:media composition remained the same as during days 6-8 (group 1), orwere changed either to EK (group 2), or to EKN (group 3), resulting in18 individual experimental conditions (FIG. 17A). Spheres cultured undereach condition were analyzed at day 9.5 by flow cytometry to quantifythe expression of PDX1 and NKX6.1, and by conventionalimmunofluorescence analysis for NKX6.1 and NEUROG3 expression. As shownin FIG. 17B, spheres within group 1 that had been exposed to retinoicacid during days 6-8, either alone or in combination with other factors(conditions 4, 5, and 6), exhibited highly efficient generation of PDX1positive progenitors (>88%), while addition of C or N alone (conditions2 and 3) did not result in enhanced generation of PDX1⁺ cells over basemedia alone. NKX6.1 was induced only weakly in all group 1 conditions,with the exception of RC (condition 5), which produced 45% PDX1/NKX6.1double positive cells. NKX6.1 expression was also strongly induced whencells were exposed to retinoic acid alone or in combination with otherfactors followed by treatment with EK (group 2) or EKN (group 3) (FIGS.17B and C, conditions 10-12 and 16-18). Endocrine differentiation,marked by NEUROG3 expression, was noted only when spheres had beenexposed to N, either between days 5-9.5 (FIG. 17C, conditions 3, 6, 9,and 12) or starting at the end of day 8 (FIG. 17C, group 3, conditions13-18). Very few NEUROG3⁺ cells were detected in all other conditions(FIG. 17C, conditions 1, 2, 4, 5, 7, 8, 10, and 11). qPCR analysis atday 8 of NEUROG3 and its downstream target NKX2.2 mRNA transcriptsrevealed significantly lower levels of these endocrine markers with Rtreatment when compared to the commonly employed RCN condition (FIG.16D). Notably, addition of vitamin C, recently shown to reduce endocrinedifferentiation in hESCs (Rezania et al, 2014), did not significantlylower NGN3 or NKX2.2 transcripts in the suspension culture system duringRCN or R treatment (FIG. 16D). Taken together, these results indicatethat R followed by EK treatment leads to highly efficient generation ofPDX1⁺/NKX6.1⁺ progenitors (90%) and that the formation of bona fideNEUROG3 positive endocrine precursors is induced by treatment with N(FIG. 17 A-C, condition 10, green gates). Thus, by defining the temporalactivities of individual signaling factors alone and in combination,transcription factor expression patterns can be induced that arecharacteristic of different human embryonic pancreatic progenitor cellstypes (PDX1⁺ and PDX1⁺/NKX6.1⁺ progenitors) without precocious inductionof endocrine differentiation.

Example 16 Recapitulating Human Pancreas Organogenesis to GenerateEndocrine Progenitors

This improved and simplified differentiation protocol provides the basisfor subsequent efficient formation of insulin-producing cells insuspension (FIG. 18A). Endocrine differentiation in PDX1/NKX6.1 doublepositive cells was induced by exposure to a cocktail of factorsconsisting of TBP (T), ALK inhibitor (A), N, and K, (TANK) which havepreviously been shown to activate NEUROG3 expression while maintaininghigh expression of PDX1 and NKX6.1 (Rezania et al, 2012; Nostro et al,2011) (FIGS. 18A and B). While NEUROG3 protein was undetectable beforeTANK treatment (FIG. 18C, day 9), cells exhibiting nuclear accumulationof NEUROG3 protein appeared as early as one day following TANK treatment(FIG. 18C, day 10). Thus, expression of the pro-endocrine factor NEUROG3is rapidly induced through TANK treatment once PDX1⁺/NKX6.1⁺ progenitorsare specified (FIG. 18B, day 9). In contrast to the near-uniformgeneration of PDX1⁺ and PDX1⁺/NKX6.1⁺ progenitor populations followingappropriate stimulation, endocrine differentiation appears to beconfined to a smaller population of cells. This observation can beexplained by the very short half-life of the NEUROG3 protein (Roark etal, 2012), which allows only transient detection of this marker in cellsundergoing endocrine differentiation. NEUROG3⁺ cells, however, continuedto be present when clusters were exposed to the endocrinedifferentiation cocktail for 5 days (FIG. 18C, day 14), indicating thatendocrine cells were being generated throughout this period. To furthercharacterize the progenitors present in the cultures at the initiationof endocrine differentiation, expression of NKX2.2, a downstream targetof NEUROG3, was analyzed. NKX2.2 has recently been reported to havedistinct expression patterns during pancreatic organogenesis in mouseand human (Jennings et al, 2013). While NKX2.2 is readily detectable inmouse pancreatic progenitor cells before NEUROG3 expression, NKX2.2protein is only observed after endocrine commitment during humanpancreas development. Similarly, NKX2.2 protein expression was detectedonly after endocrine differentiation was initiated at day 10, but notbefore in either PDX1⁺ or PDX1⁺/NKX6.1⁺ progenitors (FIG. 18C). Of note,some NKX2.2⁺ cells at day 10 co-express NEUROG3, and increasing numbersof NKX2.2⁺/NEUROG3⁻ cells are found at later time points (FIG. 18C).These data indicate that NKX2.2 could serve as a lineage tracer forhuman cells that have undergone endocrine differentiation induced bytransient NEUROG3 expression. In summary, a novel differentiationstrategy has been established that faithfully recapitulates humanpancreas organogenesis and allows for the precise control over thegeneration of PDX1⁺ and PDX1⁺/NKX6.1⁺ progenitors.

Example 17 Efficient Generation of PDX1+/NKX6.1+ Pancreatic ProgenitorCells Prior to Endocrine Induction Results in Glucose ResponsivePancreatic Beta Cells

To test whether correctly specified PDX1⁺/NKX6.1⁺ progenitor cellsundergo differentiation towards INS⁺/NKX6.1⁺ double positive pancreaticbeta cells, spheres differentiated using the new method described hereinwere transferred into a basal media without additional growth factorsand the establishment of pancreatic beta cells was monitored (FIG. 19A).The percentage of GFP⁺ cells increased from day 13 to day 19, reachingan average of 23±6% human C-peptide-positive cells at days 19-21, likelyreflecting continuous generation of insulin-producing cells for about 4days after removal of NEUROG3-inducing factors (FIG. 19B, C)Immunofluorescence analysis of insulin-producing cells revealedco-expression and nuclear localization of TFs critical for pancreaticbeta cell function (PDX1, NKX6.1 and NKX2.2), but very few polyhormonalcells (FIG. 19D). Flow cytometry analysis of differentiated clustersshowed a high percentage of total cells (black gates) and C-peptidepositive pancreatic beta cells (green gates) co-staining for PDX1,NKX6.1, NXK2.2, ISL1, PAX6, NeuroD1, and Chromogranin A (CHGA) (FIG.19E). These markers are normally found in both pancreatic progenitorsand mature pancreatic beta cells. Quantification of C-peptide pancreaticbeta cells co-staining for PDX1, NKX6.1, NKX2.2, ISL1, NEUROD1, PAX6 andChromogranin A showed 84±7%, 75±20%, 92±5%, 86±5%, 95±4%, 93±5%, and93±4% double positive cells, respectively (FIG. 19F). Notably, only 3.2%of all differentiated cells co-expressed C-peptide and the hormoneglucagon (FIG. 19E, red gate). An important hallmark of mature humanpancreatic beta cells is their restricted proliferative capacity. While9.1±3.7% of C-peptide-negative cells were actively proliferating, only0.5±0.6% of C-peptide-positive pancreatic beta cells co-stain for theproliferation marker Ki67, indicating their terminal differentiationstate (FIGS. 20A and B). Thus, the improved differentiation strategydescribed herein results in the predominant generation of post-mitotic,insulin-producing pancreatic beta cells that co-express criticalpancreatic beta cell markers.

To further characterize gene expression in pancreatic beta cells at days19-20, advantage was taken of the GFP live marker to compare sorted GFP⁺pancreatic beta cells and GFP-populations to purified human islets.hESC-derived pancreatic beta cells showed high levels of insulin genetranscripts, comparable to cadaveric islet preparations, whileGFP-negative populations exhibit only insignificant levels of thehormone (FIG. 21A). Transcript levels for two other hormones (GCG andSST) were also detected in GFP cells, likely due to contamination by thesmall number of polyhormonal cells also expressing the GFP reporter(FIGS. 21A and 5D and E). Consistent with the immunofluorescenceanalysis (FIG. 19D), transcripts for the TFs PDX1, NKX6.1 and NKX2.2normally found in both progenitor and mature pancreatic beta cells wereexpressed at comparable levels in GFP-negative, GFP-positive, and isletcells (FIG. 21B). Transcripts for the mature human pancreatic beta celltranscription factors MAFA and MAFB were robustly expressed in humanislets and enriched in pancreatic beta cells compared toGFP-populations. MAFB transcript levels in pancreatic beta cells weresimilar to human islets; MAFA expression levels were slightly lower(FIG. 21B). Other genes important for human pancreatic beta cellfunctionality, including the KATP channel components PotassiumInwardly-Rectifying Channel, Subfamily J, Member 11 (KIR6.2 also knownas KCJN11) and ATP-Binding Cassette, Sub-Family C, Member 8 (SUR1 alsoknown as ABCC8), the glucose metabolism enzyme Glucokinase (GCK, alsoknown as HK4), and the Prohormone Convertase 1/3 (PC1/3) necessary forinsulin biosynthesis, were enriched in GFP-positive pancreatic betacells at levels similar to or exceeding those found in human islets(FIG. 21C). In contrast, mRNA levels for the progenitor marker SOX9 werereduced in pancreatic beta cells compared to GFP-progenitors (FIG. 21D).The somewhat higher SOX9 expression in human islets is likely the resultof contamination with Sox9-positive duct cells. Thus, gene expressionanalysis indicated that hESC-derived pancreatic beta cells possess themolecular machinery necessary for pancreatic beta cell function,including insulin biosynthesis and glucose metabolism. Furtherinvestigations revealed that day 19 pancreatic beta cells contain2.5±1.2 ug, 0.32±0.12 ug, and 310±143 ng insulin, human c-peptide, andproinsulin per μg DNA, respectively (FIG. 21E). These values arecomparable to about 2.8 ug insulin, about 0.55 ug c-peptide, and about150 ng proinsulin per μg DNA for human islets, as recently published(Rezania et al, 2014). Western blot analysis for proinsulin and matureinsulin further confirmed efficient insulin protein processing inhESC-derived pancreatic beta cells, reaching 59±2% of the extent ofprocessing observed in purified human islets (FIGS. 22A and B).Ultrastructural analysis of differentiated cell clusters by transmissionelectron microscopy revealed that many cells contained secretoryvesicles exhibiting electron dense cores or rod-like structures, akin towhat is observed in human pancreatic beta cells (FIG. 21F). To furtherinvestigate the functional properties of in vitro differentiatedpancreatic beta cells, glucose stimulated insulin secretion assays wereperformed, in which the release of human C-peptide, a by-product ofendogenous insulin biosynthesis secreted in an equimolar ratio toinsulin, was measured. hESC-derived pancreatic beta cells analyzed atdays 19-21 responded to an increase in glucose concentration from 2.8 mMto 16.7 mM by secreting 1.8±0.9-fold more C-peptide, a response similarto the 1.9±0.6-fold increase detected with human islets (FIG. 21G).Thus, pancreatic beta cells generated by the improved differentiationstrategy described herein express critical pancreatic beta cell genes,synthesize high levels of mature insulin, exhibit ultrastructuralfeatures of bona fide pancreatic beta cells and secrete endogenousinsulin in response to changes in physiological concentrations ofglucose.

Example 18

hESC-Derived Pancreatic Beta Cells Remain Glucose Responsive after ShortTerm Transplantation

To determine whether hESC-derived pancreatic beta cells can maintaintheir glucose responsiveness in vivo, approximately 5 million cells weretransplanted under the kidney capsule of immunodeficient mice (days19-21 spheres consisting of progenitors and pancreatic beta cells). Micetransplanted with 4000 human islets served as controls. Seven to 10 dayspost-surgery, human C-peptide levels were measured in overnight-fastedmice, before and after the administration of a glucose bolus. Asexpected, mice that received human islet grafts exhibited low levels ofinsulin secretion upon fasting, followed by a marked increase incirculating insulin after glucose challenge (average of 221±116 pM, FIG.23A) Similar to mice carrying human islets, fasted mice transplantedwith hESC-derived pancreatic beta cells had low levels of circulatingC-peptide. Upon glucose administration, C-peptide concentrations in seraof these mice also increased, albeit at lower levels than in micetransplanted with human islets (average of 40±28 pM, FIG. 23A). Thislower number might be explained in part by the different numbers ofcells transplanted in the human islet and pancreatic beta cell groups.Indeed, each human islet contains on average 1000 cells, of which 50%are pancreatic beta cells (Cabrera et al, 2006). Thus, 4000 human isletscontain approximately 2.0×10⁶ bona fide pancreatic beta cells. BecausehESC differentiated spheres contain on average 23% pancreatic betacells, only about 1.15×10⁶ pancreatic beta cells were transplanted permouse. Normalization based on pancreatic beta cell number indicates thathESC derived pancreatic beta cells secreted 70±48 pM human c-peptide per2.0×10⁶ cells, representing approximately ⅓ of the insulin secreted fromeach human cadaveric pancreatic beta cell (FIG. 23A). Hematoxylin andEosin staining, together with immunofluorescence analysis of the hESCgrafts at 2 weeks post-transplantation demonstrated prominent islet-likestructures positive for human C-peptide (FIGS. 23B and C). Pancreaticbeta cells also maintained co-expression of the key pancreatic beta cellTFs PDX1, NKX6.1 and NKX2.2, and only a few cells co-expressed otherhormones, such as glucagon and somatostatin (FIG. 23C). To furtherinvestigate the functional properties of hES derived pancreatic betacells in vivo, clusters were transplanted under the kidney capsule ofmice rendered diabetic through treatment with the pancreatic beta celltoxin streptozotocin. Mice that received grafts exhibit significantlyreduced blood glucose (BG) levels at all time points analyzed whencompared to control animals (FIG. 23D). While BG levels weresignificantly reduced in graft-bearing mice, they continued to exhibithyperglycemic BG values over time. This is likely due to the limitednumber of pancreatic beta cells that can be transplanted under thekidney capsule in one mouse. It has previously been shown that 4,000human islets are required to establish long-term euglycemia in diabeticmice. Transplantation of a smaller number of human islets (1,500 islets)reduces blood glucose levels only for 7 days post-transplantation, afterwhich hyperglycemia returned (Fiaschi-Taesch et al, 2010). The surgicalprocedure described herein permits the transplantation of about 1.15×10⁶pancreatic beta cells, substantially less than the approximately 2.0×10⁶pancreatic beta cells present in the 4,000 human islets previously foundto be required for the long-term reversal of diabetes. Hence, theobserved reduction in BG levels, but lack of complete diabetes reversalin mice bearing hES-derived transplants, is not unexpected given thistechnical constraint. Taken together, the in vivo data demonstrate thathESC derived pancreatic beta cells maintain their differentiatedphenotype and remain glucose responsive after a short engraftment periodin vivo and highlights their potential therapeutic value.

Example 19

Efficient and Cost-Effective Production of Glucose Responsive PancreaticBeta Cells from Pluripotent Stem Cells

Variations on the direct differentiation method disclosed herein (see,e.g., Examples 13 and 17) were also developed that yielded efficient andcost-effective production of functional pancreatic beta cells frompluripotent stem cells. Illustrated schematically in FIG. 24, one methodaccording to the disclosure provides embryonic stem cells (e.g., humanembryonic stem cells) that were exposed to retinoic acid and incubatedfor a sufficient time to see expression of PDX1. During this time, thecells were not exposed to bone morphogenetic protein (BMP), which canlead to precocious endocrine differentiation and, ultimately, theproduction of non-functional polyhormonal cells. PDX1⁺ cells were thenexposed to 50 ng/ml epidermal growth factor (EGF) and 50 ng/mlkeratinocyte growth factor (KGF or K) in DMEM basal media containing 1XGlutaMAX™ (Invitrogen), 1X non-essential amino acids (Invitrogen) andserum-free 1X B27supplement (Life Technologies) for an extended periodof 24-48 hours under the culture conditions disclosed herein. Theextended incubation of pancreatic progenitor PDX1⁺ cells in the absenceof BMP led to the efficient production of PDX1⁺/NKX6.1⁺ pancreaticprogenitor cells. The PDX1⁺/NKX6.1⁺ cells were then incubated in anoptimized combination of growth factors to induce NGN3 expression whilemaintaining expression of PD1 and NKX6.1. The combination of factorsused in the media work in combination to yield an effective method ofdirect differentiation of pluripotent stem cells to functionalpancreatic beta cells. For example, gamma secretase inhibitor XX inducesendocrine differentiation efficiently, but does not maintain anyprogenitor expression. In combination with the other factors disclosedherein, however, its presence is tolerated and progenitor expression ismaintained. Specifically, the combination of factors includes 2 mMGlutamine (1X GlutaMAX™ (Invitrogen), 10 μg/ml heparin, 1 mM cysteine,10 μM zinc, 10 μM ALK inhibitor (Axxora), 0.5 uM BMP inhibitor (i.e.,small molecule LDN-193189; Stemgent), 1 μM T3 (triiodothyronine), and 1μM gamma secretase inhibitor XX. It is expected that the combination offactors is important in the gradual elaboration of endocrine functionwhile retaining characteristics of progenitor cells. The results of thedirect differentiation method as performed on embryonic stem cells isthe efficient, direct differentiation of such cells into functional(glucose-responsive) pancreatic beta cells, as shown in FIG. 25.Alternative media and culture conditions are also compatible with thedirect differentiation methods disclosed herein, as evidenced by theculture media used in the differentiation protocol described in Example13 under Cell Culture.

In addition, the culture media in which the PDX1⁺/NKX6.1⁺ pancreaticprogenitor cells were incubated was supplemented with vitamin C andBayK-8644. The addition of these two compounds led to the production ofpancreatic beta cells of robust functionality. In particular, asdescribed in Example 18, the pancreatic beta cells areglucose-responsive and remain responsive after transplantation.

The disclosure provides embodiments of the direct differentiationmethods in which the PDX1⁺ pancreatic progenitor cell is exposed to aneffective amount of either EGF or KGF, as well as the above-describedmethod in which the cells were exposed to effective amounts (e.g., 50ng/ml) of both EGF and KGF. Additional experiments established that themethods of direct differentiation could involve incubation of cells inculture media containing EGF and/or KGF for up to 32 days. Accordingly,the disclosure contemplates embodiments in which the PDX1⁺ cells areincubated in the presence of EGF and/or KGF for any time period between24 hours and 32 days.

Thus, the direct differentiation methods disclosed herein that includevitamin C and BayK-8644 provide efficient, effective, quick andinexpensive approaches to differentiating pluripotent stem cells intofunctional pancreatic beta cells.

Example 20

Production of Glucose Responsive Pancreatic Beta Cells from PluripotentStem Cells Under Physiological Oxygen Levels

Additional experimentation has established that embodiments of thedisclosed direct differentiation methods yield functional pancreaticbeta cells following the protocol disclosed herein (see, e.g., Examples13, 17 and 19), but with the additional feature that the cells areexposed to lower levels of oxygen, closer to the physiological situationfound in vivo. The data show that pancreatic beta cells can be, andwere, generated under reduced oxygen levels (5%) closer to normalphysiological levels of about 4-5%. FIG. 27. The loss of functionalproperties upon reduction to physiological O₂ levels (approximately4-5%) from ambient levels (approximately 20%) that had been observedwith other approaches to the production of pancreatic beta cells is acritical shortcoming for cell therapy and transplantation. Cellscultured at ambient oxygen levels experience a loss of pancreatic betacell features upon transplantation into recipients, where they areimmediately exposed to physiological oxygen levels. Indeed, the dataindicate that while some of the transplanted pancreatic beta cellsmaintain a functional phenotype, many lose essential functional featuresor undergo cell death. This reduction in functionality is connected to aloss of the differentiated phenotype and to a loss of the identity ofthe pancreatic beta cells. The disclosure herein of methods for thedirect differentiation of pluripotent stem cells (e.g., embryonic stemcells), wherein the cells have been adapted to lower O₂ concentrationseither during the pluripotent state or after generation of pancreaticprogenitor cells, provides an advance over the state of the art inyielding a method for producing a cell therapy product in the form of afunctional pancreatic beta cell useful for the treatment of diabetes bymaintaining a functional pancreatic beta cell phenotype upontransplantation (FIG. 26).

Each of the references listed below and cited throughout the disclosureis incorporated by reference herein in its entirety, or in relevantpart, as would be apparent from context.

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The disclosed subject matter has been described with reference tovarious specific embodiments and techniques. It should be understood,however, that many variations and modifications may be made whileremaining within the spirit and scope of the disclosed subject matter.

All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby specifically incorporated by reference to the same extent asif it had been incorporated by reference in its entirety individually orset forth herein in its entirety. Applicants reserve the right tophysically incorporate into this specification any and all materials andinformation from any such cited patents or publications.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and the methods and processes are not necessarilyrestricted to the orders of steps indicated herein or in the claims.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims and statements of theinvention. Under no circumstances may the patent be interpreted to belimited to the specific examples or embodiments or methods specificallydisclosed herein. Under no circumstances may the patent be interpretedto be limited by any statement made by any Examiner or any otherofficial or employee of the Patent and Trademark Office unless suchstatement is specifically and without qualification or reservationexpressly adopted in a responsive writing by Applicants.

What is claimed:
 1. A method comprising: a. contacting pancreaticendodermal progenitor cells with a composition comprising vitamin C,BayK-8644, heparin, zinc sulfate, Alk5 inhibitor II, LDN-193189, T3thyroid hormone, and Compound E for three to fifteen days to generate afirst population of cells; b. contacting the first population of cellswith a second composition comprising vitamin C, BayK-8644, heparin, zincsulfate, Alk5 inhibitor II, T3 thyroid hormone, cysteine, Trolox, andR428 for three to fifteen days to generate a second population of cells;and c. culturing the second population of cells as 3D aggregates inlow-attachment plates for three to fifteen days to thereby generate athird cell population comprising functional pancreatic beta-like cells.2. The method of claim 1, further comprising administering thefunctional pancreatic beta-like cells to a mammal in need thereof. 3.The method of claim 2, wherein the mammal in need thereof has type Idiabetes, type II diabetes, or type 1.5 diabetes.
 4. The method of claim2 or claim 3, wherein the mammal is a human.
 5. A method of producing apancreatic beta cell from a stem cell comprising: (a) exposing a stemcell to Epidermal Growth Factor and/or Keratinocyte Growth Factor for12-72 hours under conditions suitable for cell culture growth, therebymaintaining a progenitor cell; (b) incubating the progenitor cell in aculture medium comprising heparin, cysteine, zinc, ALK inhibitor, BMPinhibitor LDN-193189, T3 thyroid hormone and gamma secretase inhibitorXX to yield a cell in culture; and (c) adding to the cell in culturevitamin C and BayK-8644, thereby producing a functional pancreatic betacell.
 6. The method of claim 5, wherein the stem cell of step (a) isexposed to 10-300 ng/ml Epidermal Growth Factor or 10-300 ng/ml ofKeratinocyte Growth Factor; the culture medium of step (b) comprises2-20 μg/ml heparin, 0.2-5 mM cysteine, 2-20 μM zinc, 2-20 μM ALKinhibitor, 0.2-2 μM BMP inhibitor LDN-193189, 0.2-5 μM T3 thyroidhormone and 0.2-5 μM gamma secretase inhibitor XX; and step (c)comprises adding to the cell in culture 10-2000 μM vitamin C and 0.2-5μM BayK-8644.
 7. The method of claim 6, wherein the stem cell of step(a) is exposed to 50 ng/ml Epidermal Growth Factor or 50 ng/ml ofKeratinocyte Growth Factor; the culture medium of step (b) comprises 10μg/ml heparin, 1 mM cysteine, 10 μM zinc, 10 μM ALK inhibitor, 0.5 μMBMP inhibitor LDN-193189, 1 μM T3 thyroid hormone and 1 μM gammasecretase inhibitor XX; and step (c) comprises adding to the cell inculture 500 μM vitamin C and 2 μM BayK-8644.
 8. The method of any one ofclaims 5-7, wherein said exposing of step (a) is in DMEM comprising0.1-5 mM glutamine or 0.05-2.5X GlutaMAX™, 0.1-5X non-essential aminoacids, and 0.1-5X B27 supplement.
 9. The method of any one of claims5-8, wherein the culture medium of step (b) comprises DMEM comprising0.1-5 mM glutamine or 0.05-2.5X GlutaMAX™, and 0.1-5X non-essentialamino acids.
 10. The method of any one of claims 5-9, wherein saidexposing of step (a) is in DMEM comprising 2 mM glutamine or 1XGlutaMAX™.
 11. The method of any one of claims 5-10, wherein the culturemedium of step (b) comprises DMEM comprising 2 mM glutamine or 1XGlutaMAX™.
 12. The method of any one of claims 5-11, wherein the stemcell is exposed to Epidermal Growth Factor or Keratinocyte Growth Factorfor 24-48 hours.
 13. The method of any one of claims 5-12, wherein thestem cell is an embryonic stem cell.
 14. The method of any one of claims5-13, wherein the stem cell is a human stem cell.
 15. The method of anyone of claims 5-14, wherein the stem cell is exposed to epidermal growthfactor.
 16. The method of any one of claims 5-15, wherein the stem cellis exposed to keratinocyte growth factor.
 17. The method of any one ofclaims 5-14, wherein the stem cell is exposed to epidermal growth factorand keratinocyte growth factor.
 18. The method of any one of claims5-17, wherein the functional pancreatic beta cell exhibits a 1-7-foldincrease in insulin secretion upon stimulation with glucose.
 19. Themethod of any one of claims 5-17, wherein the functional pancreatic betacell exhibits a 2-fold or more increase in insulin secretion uponstimulation with glucose.
 20. The method of any one of claims 5-19,wherein the pancreatic beta cell is functional immediately upontransplantation.
 21. The method of any one of claims 5-20, wherein thepancreatic beta cell is functional within one week of transplantation.22. The method of any one of claims 5-21, wherein the pancreatic betacell remains functional for at least four weeks.
 23. The method of anyone of claims 5-22, further comprising exposure of the stem cell to anoxygen level no greater than 10% O₂.
 24. The method of claim 23, whereinthe oxygen level is no greater than 5% O₂.
 25. The method of claim 24,wherein the oxygen level is no greater than 4% O₂.
 26. The method of anyone of claims 5-25, wherein NGN3 and PDX1/NKX6.1 are expressed duringthe incubating step and the adding step.
 27. The method of any one ofclaims 5-26, wherein at least 75% of the stem cells differentiate intofunctional pancreatic beta cells.
 28. The method of any one of claims5-27, wherein the progenitor cell is maintained for up to 32 days inculture containing Epidermal Growth Factor or Keratinocyte GrowthFactor.
 29. A functional pancreatic beta cell produced according to themethod of any one of claims 5-28.
 30. The functional pancreatic betacell of claim 29, wherein the cell is a human cell.
 31. The functionalpancreatic beta cell of claim 29 or claim 30, wherein the cell expressesat least three pancreatic cell markers selected from the group of humanc-peptide (C-PEP), Chromagranin A (CHGA), transcription factor NKX6.1,transcription factor PDX1, transcription factor PAX6, transcriptionfactor NKX2.2, transcription factor NEUROD1 and transcription factorISL1 without inducing expression of Glucagon (GCG) or Somatostatin(SST).
 32. A method for treating diabetes comprising administering aneffective amount of the cell according to any one of claims 29-31, to adiabetic subject.
 33. The method of claim 32, wherein the subject ishuman.